Methods for reducing noise in signal-generating digital assays

ABSTRACT

Methods of reducing background noise in signal-generating digital assays, such as fluorescent digital immunoassays, using a colorant are disclosed. The methods utilize a binding member that specifically binds to an analyte in a biological sample. The binding member is conjugated to a signal generating compound, e.g., an enzyme, which is dissociated from the binding member. A colorant is added with a signal generating substrate, e.g., a fluorogenic or chromogenic substrate for the enzyme, or after a signal generating substrate to reduce background noise in a fluorescent digital immunoassay. The signal generated between the signal generating compound and the signal generating substrate is detected and correlated to the presence and/or concentration of the analyte.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application is a continuation of granted U.S. application Ser. No.15/888,952, filed Feb. 5, 2018, now U.S. Pat. No. 11,047,854, issuedJun. 29, 2021, which claims the benefit of U.S. Provisional PatentApplication No. 62/455,436, filed on Feb. 6, 2017, both of which areincorporated by reference herein.

TECHNICAL FIELD

This disclosure relates to methods of reducing background noise insignal-generating digital assays using a colorant, such as an ink ordye.

BACKGROUND

Methods and devices that can accurately analyze analyte(s) of interestin a sample are essential for diagnostics, such as for examplediagnosing a disease, disorder or condition, prognostics, environmentalassessment, food safety, detection of chemical or biological warfareagents and the like. Most current techniques for quantifying low levelsof analyte molecules in a sample use amplification procedures toincrease the number of reporter molecules to provide a measurablesignal. Examples of current techniques include enzyme-linkedimmunosorbent assays (ELISA) for amplifying the signal in antibody-basedassays, as well as the polymerase chain reaction (PCR) for amplifyingtarget DNA strands in DNA-based assays. Most detection schemes requirethe presence of a large number of molecules in the ensemble for theaggregate signal to be above the detection threshold. This requirementlimits the sensitivity of most detection techniques and the dynamicrange (i.e., the range of concentrations that can be detected). Many ofthe known methods and techniques are further plagued with problems ofnon-specific binding, which leads to an increase in the backgroundsignal and limits the lowest concentration that may be accurately orreproducibly detected.

Digital ELISA is a candidate for the next generation of immunoassays asit can detect one molecule of enzyme using a conjugate. See FIGS. 1 and2. In the digital ELISA procedure, individual target analytes arecaptured on antibody-coated solid phases (e.g., beads) and then reactedwith detection antibody conjugated with an enzyme. After removingunbound detection antibody, the beads are entrapped in each dropletchamber with the substrate of the enzyme and the beads are enclosed withthe heavy oil (see yellow layer in step 3 of FIG. 2). During steps 3-8of FIG. 2, the aqueous phase is displaced by the heavy oil andsubsequently removed. It typically takes 30-40 minutes to completelyremove the aqueous phase, which also includes the fluorescent substance(see sky blue layer in steps 1-7 of FIG. 2. Because the removal step 7shown in FIG. 2 is performed manually, this poses challenges given thewide variety of times such assays are performed which results inaccuracy issues depending upon the individual differences of each assayas well as the number of samples being tested. By way of example, ifstep 7 is performed incorrectly (i.e., there is incomplete removal ofthe aqueous layer), the signal (“glow”) background noise obstructs thecounting of the number of fluorescent droplets under a digital countingdevice (e.g., an optical microscope). In addition, any light source,such as a room lamp, may cause high background noise. As a result, thereis a need for improved digital assays that are easily adaptable for ahigh throughput assay with reduced background noise, i.e., backgroundsignal (e.g., fluorescence or glow), and high accuracy.

SUMMARY

The present invention is directed to a signal-generating digital assayfor determining the presence or absence of a single molecule of ananalyte in a fluid sample. The assay includes: (a) contacting the fluidsample containing or suspected of containing the analyte with aplurality of solid supports to create a mixture, wherein each solidsupport comprises one or more first specific binding member capable ofbinding to the analyte thereby producing solid support-first specificbinding member-analyte complexes; (b) washing the solid support-firstspecific binding member-analyte complexes in the mixture with a firstwash buffer to remove any solid support-first specific binding membernot bound to the analyte thereby producing a washed mixture; (c) addingto the washed mixture one or more second specific binding memberscapable of binding to the analyte thereby producing solid support-firstspecific binding member-analyte-second specific binding membercomplexes, wherein the second specific binding member comprises a signalgenerating compound attached thereto; (d) washing the solidsupport-first specific binding member-analyte-second specific bindingmember complexes with a second wash buffer to remove any second specificbinding member not bound to the analyte bound to the first specificbinding member thereby producing washed solid support complexes; (e)adding to the washed solid support complexes a signal generatingsubstrate and a colorant, wherein the signal generating compound and thesignal generating substrate produce a detectable signal; (f) spatiallysegregating at least a portion of the washed solid support complexesinto a plurality of separate locations, wherein the plurality ofseparation locations comprises a plurality of reaction vessels andwherein the washed solid support complexes are present in an aqueousphase; (g) covering the plurality of reaction vessels with a sealant,wherein the aqueous phase is displaced by the sealant in the reactionchamber; and (h) detecting the presence or absence of the detectablesignal using a digital counting device, wherein detection of thepresence of the detectable signal indicates the presence of a singlemolecule of analyte in the sample.

The present invention is directed to a fluorescent digital immunoassayfor determining the presence or absence of a single molecule of ananalyte in a fluid sample. The immunoassay includes: (a) contacting thefluid sample containing or suspected of containing the analyte with aplurality of solid supports to create a mixture, wherein each solidsupport comprises one or more first specific binding member capable ofbinding to the analyte thereby producing solid support-first specificbinding member-analyte complexes; (b) washing the solid support-firstspecific binding member-analyte complexes in the mixture with a firstwash buffer to remove any solid support-first specific binding membernot bound to the analyte thereby producing a washed mixture; (c) addingto the washed mixture one or more second specific binding memberscapable of binding to the analyte thereby producing solid support-firstspecific binding member-analyte-second specific binding membercomplexes, wherein the second specific binding member comprises a signalgenerating compound attached thereto; (d) washing the solidsupport-first specific binding member-analyte-second specific bindingmember complexes with a second wash buffer to remove any second specificbinding member not bound to the analyte bound to the first specificbinding member thereby producing washed solid support complexes; (e)adding to the washed solid support complexes a signal generatingsubstrate and a colorant, wherein the signal generating compound and thesignal generating substrate produce a detectable signal; (f) spatiallysegregating at least a portion of the washed solid support complexesinto a plurality of separate locations, wherein the plurality ofseparation locations comprises a plurality of reaction vessels andwherein the washed solid support complexes are present in an aqueousphase; (g) covering the plurality of reaction vessels with a sealant,wherein the aqueous phase is displaced by the sealant in the reactionchamber; and (h) detecting the presence or absence of the detectablesignal using a digital counting device, wherein detection of thepresence of the detectable signal indicates the presence of a singlemolecule of analyte in the sample.

The present invention is directed to a signal-generating digital assayfor determining the presence or absence of a single molecule of ananalyte in a fluid sample. The assay includes: (a) contacting the fluidsample containing or suspected of containing the analyte with aplurality of solid supports to create a mixture, wherein each solidsupport comprises one or more first specific binding member capable ofbinding to the analyte thereby producing solid support-first specificbinding member-analyte complexes; (b) washing the solid support-firstspecific binding member-analyte complexes in the mixture with a firstwash buffer to remove any solid support-first specific binding membernot bound to the analyte thereby producing a washed mixture; (c) addingto the washed mixture one or more second specific binding memberscapable of binding to the analyte thereby producing solid support-firstspecific binding member-analyte-second specific binding membercomplexes, wherein the second specific binding member comprises a signalgenerating compound attached thereto; (d) washing the solidsupport-first specific binding member-analyte-second specific bindingmember complexes with a second wash buffer to remove any second specificbinding member not bound to the analyte bound to the first specificbinding member thereby producing washed solid support complexes; (e)adding to the washed solid support complexes a signal generatingsubstrate, wherein the signal generating compound and the signalgenerating substrate produce a detectable signal; (f) spatiallysegregating at least a portion of the washed solid support complexesinto a plurality of separate locations, wherein the plurality ofseparation locations comprises a plurality of reaction vessels andwherein the washed solid support complexes are present in an aqueousphase; (g) covering the plurality of reaction vessels with a sealant,wherein the aqueous phase is displaced by the sealant in the reactionchamber; (h) adding a colorant to the displaced aqueous phase; and (i)detecting the presence or absence of the detectable signal using adigital counting device, wherein detection of the presence of thedetectable signal indicates the presence of a single molecule of analytein the sample.

The present invention is directed to a signal-generating digital assayfor determining the presence or absence of a single molecule of ananalyte in a fluid sample. The assay includes: (a) contacting the fluidsample containing or suspected of containing the analyte with aplurality of solid supports to create a mixture, wherein each solidsupport comprises one or more first specific binding member capable ofbinding to the analyte thereby producing solid support-first specificbinding member-analyte complexes; (b) washing the solid support-firstspecific binding member-analyte complexes in the mixture with a firstwash buffer to remove any solid support-first specific binding membernot bound to the analyte thereby producing a washed mixture; (c) addingto the washed mixture one or more second specific binding memberscapable of binding to the analyte thereby producing solid support-firstspecific binding member-analyte-second specific binding membercomplexes, wherein the second specific binding member comprises a signalgenerating compound attached thereto; (d) washing the solidsupport-first specific binding member-analyte-second specific bindingmember complexes with a second wash buffer to remove any second specificbinding member not bound to the analyte bound to the first specificbinding member thereby producing washed solid support complexes; (e)adding to the washed solid support complexes a signal generatingsubstrate, wherein the signal generating compound and the signalgenerating substrate produce a detectable signal; (f) spatiallysegregating at least a portion of the washed solid support complexesinto a plurality of separate locations, wherein the plurality ofseparation locations comprises a plurality of reaction vessels andwherein the washed solid support complexes are present in an aqueousphase; (g) covering the plurality of reaction vessels with a sealant,wherein the aqueous phase is displaced by the sealant in the reactionchamber; (h) adding a colorant to the sealant; and (i) detecting thepresence or absence of the detectable signal using a digital countingdevice, wherein detection of the presence of the detectable signalindicates the presence of a single molecule of analyte in the sample.

The present invention is directed to a fluorescent digital immunoassayfor determining the presence or absence of a single molecule of ananalyte in a fluid sample. The immunoassay includes: (a) contacting thefluid sample containing or suspected of containing the analyte with aplurality of solid supports to create a mixture, wherein each solidsupport comprises one or more first specific binding member capable ofbinding to the analyte thereby producing solid support-first specificbinding member-analyte complexes; (b) washing the solid support-firstspecific binding member-analyte complexes in the mixture with a firstwash buffer to remove any solid support-first specific binding membernot bound to the analyte thereby producing a washed mixture; (c) addingto the washed mixture one or more second specific binding memberscapable of binding to the analyte thereby producing solid support-firstspecific binding member-analyte-second specific binding membercomplexes, wherein the second specific binding member comprises a signalgenerating compound attached thereto; (d) washing the solidsupport-first specific binding member-analyte-second specific bindingmember complexes with a second wash buffer to remove any second specificbinding member not bound to the analyte bound to the first specificbinding member thereby producing washed solid support complexes; (e)adding to the washed solid support complexes a signal generatingsubstrate, wherein the signal generating compound and the signalgenerating substrate produce a detectable signal; (f) spatiallysegregating at least a portion of the washed solid support complexesinto a plurality of separate locations, wherein the plurality ofseparation locations comprises a plurality of reaction vessels andwherein the washed solid support complexes are present in an aqueousphase; (g) covering the plurality of reaction vessels with a sealant,wherein the aqueous phase is displaced by the sealant in the reactionchamber; (h) adding a colorant to the displaced aqueous phase; and (i)detecting the presence or absence of the detectable signal using adigital counting device, wherein detection of the presence of thedetectable signal indicates the presence of a single molecule of analytein the sample.

The present invention is directed to a fluorescent digital immunoassayfor determining the presence or absence of a single molecule of ananalyte in a fluid sample. The immunoassay includes: (a) contacting thefluid sample containing or suspected of containing the analyte with aplurality of solid supports to create a mixture, wherein each solidsupport comprises one or more first specific binding member capable ofbinding to the analyte thereby producing solid support-first specificbinding member-analyte complexes; (b) washing the solid support-firstspecific binding member-analyte complexes in the mixture with a firstwash buffer to remove any solid support-first specific binding membernot bound to the analyte thereby producing a washed mixture; (c) addingto the washed mixture one or more second specific binding memberscapable of binding to the analyte thereby producing solid support-firstspecific binding member-analyte-second specific binding membercomplexes, wherein the second specific binding member comprises a signalgenerating compound attached thereto; (d) washing the solidsupport-first specific binding member-analyte-second specific bindingmember complexes with a second wash buffer to remove any second specificbinding member not bound to the analyte bound to the first specificbinding member thereby producing washed solid support complexes; (e)adding to the washed solid support complexes a signal generatingsubstrate, wherein the signal generating compound and the signalgenerating substrate produce a detectable signal; (f) spatiallysegregating at least a portion of the washed solid support complexesinto a plurality of separate locations, wherein the plurality ofseparation locations comprises a plurality of reaction vessels andwherein the washed solid support complexes are present in an aqueousphase; (g) covering the plurality of reaction vessels with a sealant,wherein the aqueous phase is displaced by the sealant in the reactionchamber; (h) adding a colorant to the sealant; and (i) detecting thepresence or absence of the detectable signal using a digital countingdevice, wherein detection of the presence of the detectable signalindicates the presence of a single molecule of analyte in the sample.

The present invention is directed to a method for reducing fluorescencebackground noise in a signal-generating digital assay used to detect ananalyte in a sample. The method includes: (a) contacting a fluid samplecontaining or suspected of containing the analyte with a plurality ofsolid supports to create a mixture, wherein each solid support comprisesone or more first specific binding member capable of binding to theanalyte thereby producing solid support-first specific bindingmember-analyte complexes; (b) washing the solid support-first specificbinding member-analyte complexes in the mixture with a first wash bufferto remove any solid support-first specific binding member not bound tothe analyte thereby producing a washed mixture; (c) adding to the washedmixture one or more second specific binding members capable of bindingto the analyte thereby producing solid support-first specific bindingmember-analyte-second specific binding member complexes, wherein thesecond specific binding member comprises a signal generating compoundattached thereto; (d) washing the solid support-first specific bindingmember-analyte-second specific binding member complexes with a secondwash buffer to remove any second specific binding member not bound tothe analyte bound to the first specific binding member thereby producingwashed solid support complexes; (e) adding to the washed solid supportcomplexes a signal generating substrate, wherein the signal generatingcompound and the signal generating substrate produce a detectablesignal; (f) spatially segregating at least a portion of the washed solidsupport complexes into a plurality of separate locations, wherein theplurality of separation locations comprises a plurality of reactionvessels and wherein the washed solid support complexes are present in anaqueous phase; (g) covering the plurality of reaction vessels with asealant, wherein the aqueous phase is displaced by the sealant in thereaction chamber; and (h) detecting the presence or absence of thedetectable signal using a digital counting device, wherein detection ofthe presence of the detectable signal indicates the presence of a singlemolecule of analyte in the sample, wherein the digital counting devicecomprises a black device.

The present invention is directed to a method for reducing fluorescencebackground noise in a fluorescent digital immunoassay used to detect ananalyte in a sample. The method includes: (a) contacting a fluid samplecontaining or suspected of containing the analyte with a plurality ofsolid supports to create a mixture, wherein each solid support comprisesone or more first specific binding member capable of binding to theanalyte thereby producing solid support-first specific bindingmember-analyte complexes; (b) washing the solid support-first specificbinding member-analyte complexes in the mixture with a first wash bufferto remove any solid support-first specific binding member not bound tothe analyte thereby producing a washed mixture; (c) adding to the washedmixture one or more second specific binding members capable of bindingto the analyte thereby producing solid support-first specific bindingmember-analyte-second specific binding member complexes, wherein thesecond specific binding member comprises a signal generating compoundattached thereto; (d) washing the solid support-first specific bindingmember-analyte-second specific binding member complexes with a secondwash buffer to remove any second specific binding member not bound tothe analyte bound to the first specific binding member thereby producingwashed solid support complexes; (e) adding to the washed solid supportcomplexes a signal generating substrate, wherein the signal generatingcompound and the signal generating substrate produce a detectablesignal; (f) spatially segregating at least a portion of the washed solidsupport complexes into a plurality of separate locations, wherein theplurality of separation locations comprises a plurality of reactionvessels and wherein the washed solid support complexes are present in anaqueous phase; (g) covering the plurality of reaction vessels with asealant, wherein the aqueous phase is displaced by the sealant in thereaction chamber; and (h) detecting the presence or absence of thedetectable signal using a digital counting device, wherein detection ofthe presence of the detectable signal indicates the presence of a singlemolecule of analyte in the sample, wherein the digital counting devicecomprises a black device.

BRIEF DESCRIPTION OF THE DRAWINGS

The details of the subject matter set forth herein, both as to itsstructure and operation, may be apparent by study of the accompanyingfigures, in which like reference numerals refer to like parts. Thecomponents in the figures are not necessarily to scale, emphasis insteadbeing placed upon illustrating the principles of the subject matter.Moreover, all illustrations are intended to convey concepts, whererelative sizes, shapes and other detailed attributes may be illustratedschematically rather than literally or precisely.

FIG. 1 shows the procedure of a fluorescent digital ELISA assay.

FIG. 2 shows a procedure of a fluorescent digital ELISA assay using adrip oil sealing step and an aqueous phase removal step.

FIG. 3 shows an improved procedure of a fluorescent digital ELISA assayusing “Black Ink Addition” to the displaced aqueous phase.

FIG. 4 shows image analysis data of treatments A (digital ELISA that didnot remove the aqueous phase) (FIG. 4A), B (digital ELISA that removedthe aqueous phase) (FIG. 4B), and C (black ink added to the aqueousphase and the aqueous phase was not removed) (FIG. 4C), using atetramethylrhodamine (TRITC) filter for counting the number of beads anda fluorescein filter for counting the number of fluorescent droplets.

FIG. 5 shows the signal % (true signal) for treatments (B) and (C) asdescribed in Example 1.

FIG. 6 shows 2 representative patterns of glowed images in fluoresceindetection (16 unanalyzable pieces of treatment (B)).

FIG. 7A shows an improved procedure of a fluorescent digital ELISA assayusing black ink mixing with the solution for enzymatic reaction.

FIG. 7B shows a schematic of how the disclosed methods can limit thelight area similar to an evanescent light detection system.

FIG. 8 shows the signal % (true signal) and S/N ratio of the “Black InkPre-Mix” study.

FIG. 9A shows an example of India Ink.

FIG. 9B shows the chemical structure of Acid black 2 (“ABk2”).

FIG. 9C shows the chemical structure of Acid Orange 7 (“AO7”).

FIG. 9D shows the chemical structure of Direct Blue 14 (“DBu14”).

FIG. 10 shows the signal % (true signal) and S/N ratio of the dyecompound pre-mix study.

FIG. 11 shows the signal % (true signal) of the dye combination study.Black Ink: India Ink, ABk2: Acid Black 2, AO7: Acid Orange 7 and DBu14:Direct Blue 14.

FIGS. 12A and 12B show the absorbing spectra of the combination of 15 mMAcid Orange 7 (AO7) and 4 mM Direct Blue 14 (DBu14) (FIG. 12A) and theemission and excitation spectra of Fluorescein (FIG. 12B). Note;emission peak is located around the valley between AO7 and DBu14.

FIG. 13 shows the signal % (true signal) and S/N ratio of the improvedfluorescent digital immunoassay, “Addition method” and “Pre-mix method,”for the detection of low concentration of antigen (0.1 fM antigensample) after 30 minutes. The antigen was recombinant hepatitis Bsurface antigen (rHBsAg).

FIG. 14 shows an alternative embodiment for reducing background noise.

DETAILED DESCRIPTION

Embodiments of the present disclosure relate to the use of a colorant,such as one or more dark/black inks or dyes, to reduce background signal(e.g., fluorescence) in an assay, such as a digital ELISA, without theneed to remove an aqueous phase. For example, in digital immunoassays,analytes of interest are captured on solid supports between a capturemolecule and a detection molecule (which is bound to a signal generatingcompound (e.g., such as an enzyme)) to form a capture molecule-analyteof interest-detection molecule complex. The solid supports containingthe capture molecule-analyte of interest-detection molecule complex arethen entrapped in a droplet and the droplet is moved to an array ofreaction vessels (e.g., wells) which creates an aqueous phase withineach of the reaction vessels. The reaction vessels can be sealed orcovered in a “solvent well-sealing” method by the addition of a sealantthat includes one or more solvents, such as a hydrophilic or ahydrophobic solvent that has a density that is heavier than the aqueousphase. After the sealant is added to the reaction vessels, the sealantmoves towards the bottom of the reaction vessel because of its heavierdensity and displaces the aqueous phase, thus forcing it to the surfaceand creating a clear separation between the upper aqueous phase and thelower solvent phase. For example, the plurality of reaction vessels canbe covered with heavy fluorinated oil and the aqueous phase and the oilphase are changed. After the aqueous phase and the oil phase arechanged, the aqueous phase is removed. The upper aqueous phase can beremoved using routine techniques known in the art.

One of the problems with the solvent sealing method is that the residualaqueous phase can cause background signal or detectable label (e.g.,fluorescence). Another source of background signal is any light, such asa room lamp. Another problem is the need to remove the aqueous solutionfrom the oil before detection, which is a very time consuming step andleads to very low throughput of the assay and very variable incubationtime for each test. The addition of a colorant to any solution phase,including the aqueous phase or sealant (e.g., oil) phase, suppresses thebackground fluorescent noise and eliminates the need to remove theaqueous phase, improving the ease with which the assay can be performedand the quality of the images obtained during analysis. Thus, thepresent invention simplifies and shortens the protocol of digital assaysby reducing the number of steps needed to perform the assay. Forexample, the need to remove the aqueous solution from the oil beforedetection is eliminated by the presently disclosed method. In addition,the present invention can be modified for use in high throughput assay,applied for full automatic dispensing system, reduce background of theassay produced signal, and improve data accuracy. The present inventioncan solve both the low throughput issue of the digital assays,particularly digital ELISA assays and the accuracy issue of the datadepending on the individual differences or the number of testing.Additionally, the improved method described herein provides a uniqueapplication that can mimic “evanescent light detection system” withoutrequiring the use of highly expensive digital counting devices such as ascanning near-field optical microscope. The presently disclosed methods,for example, the method shown in FIG. 7A, can limit the light area (asshown in FIG. 7B), which is comparable to the light area in anevanescent light detection system.

1. Definitions

Before the embodiments of the present disclosure are described, it is tobe understood that this invention is not limited to particularembodiments described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

“Comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” andvariants thereof, as used herein, are intended to be open-endedtransitional phrases, terms, or words that do not preclude thepossibility of additional acts or structures. The singular forms “a,”“and” and “the” include plural references unless the context clearlydictates otherwise. The present disclosure also contemplates otherembodiments “comprising,” “consisting of” and “consisting essentiallyof,” the embodiments or elements presented herein, whether explicitlyset forth or not.

For the recitation of numeric ranges herein, each intervening numberthere between with the same degree of precision is explicitlycontemplated. For example, for the range of 6-9, the numbers 7 and 8 arecontemplated in addition to 6 and 9, and for the range 6.0-7.0, thenumber 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 areexplicitly contemplated.

“Affinity” and “binding affinity” as used interchangeably herein referto the tendency or strength of binding of the binding member to theanalyte. For example, the binding affinity may be represented by theequilibrium dissociation constant (K_(D)), the dissociation rate(k_(d)), or the association rate (k_(a)).

“Analog” as used herein refers to a molecule that has a similarstructure to a molecule of interest (e.g., nucleoside analog, nucleotideanalog, sugar phosphate analog, analyte analog, etc.). An analyte analogis a molecule that is structurally similar to an analyte but for whichthe binding member has a different affinity.

“Analyte”, “target analyte”, “analyte of interest” as usedinterchangeably herein, refer to an analyte being measured in themethods and devices disclosed herein. Analytes of interest are furtherdescribed herein.

“Antibody” and “antibodies” as used herein refers to monoclonalantibodies, multispecific antibodies, human antibodies, humanizedantibodies (fully or partially humanized), animal antibodies such as,but not limited to, a bird (for example, a duck or a goose), a shark, awhale, and a mammal, including a non-primate (for example, a cow, a pig,a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, aguinea pig, a cat, a dog, a rat, a mouse, etc.) or a non-human primate(for example, a monkey, a chimpanzee, etc.), recombinant antibodies,chimeric antibodies, single-chain Fvs (“scFv”), single chain antibodies,single domain antibodies, Fab fragments, F(ab′) fragments, F(ab′)2fragments, disulfide-linked Fvs (“sdFv”), and anti-idiotypic (“anti-Id”)antibodies, dual-domain antibodies, dual variable domain (DVD) or triplevariable domain (TVD) antibodies (dual-variable domain immunoglobulinsand methods for making them are described in Wu, C., et al., NatureBiotechnology, 25(11):1290-1297 (2007) and PCT International PatentApplication WO 2001/058956, the contents of each of which are hereinincorporated by reference), and functionally active epitope-bindingfragments of any of the above. In particular, antibodies includeimmunoglobulin molecules and immunologically active fragments ofimmunoglobulin molecules, namely, molecules that contain ananalyte-binding site. Immunoglobulin molecules can be of any type (forexample, IgG, IgE, IgM, IgD, IgA, and IgY), class (for example, IgG1,IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. For simplicity sake, anantibody against an analyte is frequently referred to herein as beingeither an “anti-analyte antibody” or merely an “analyte antibody.”

“Antibody fragment” as used herein refers to a portion of an intactantibody comprising the antigen-binding site or variable region. Theportion does not include the constant heavy chain domains (i.e., CH2,CH3, or CH4, depending on the antibody isotype) of the Fc region of theintact antibody. Examples of antibody fragments include, but are notlimited to, Fab fragments, Fab′ fragments, Fab′-SH fragments, F(ab′)2fragments, Fd fragments, Fv fragments, diabodies, single-chain Fv (scFv)molecules, single-chain polypeptides containing only one light chainvariable domain, single-chain polypeptides containing the three CDRs ofthe light-chain variable domain, single-chain polypeptides containingonly one heavy chain variable region, and single-chain polypeptidescontaining the three CDRs of the heavy chain variable region.

“Bead” and “particle” are used herein interchangeably and refer to asubstantially spherical solid support.

“Binding Protein” is used herein to refer to a monomeric or multimericprotein that binds to and forms a complex with a binding partner, suchas, for example, a polypeptide, an antigen, a chemical compound or othermolecule, or a substrate of any kind. A binding protein specificallybinds a binding partner. Binding proteins include antibodies, as well asantigen-binding fragments thereof and other various forms andderivatives thereof as are known in the art and described herein below,and other molecules comprising one or more antigen-binding domains thatbind to an antigen molecule or a particular site (epitope) on theantigen molecule. Accordingly, a binding protein includes, but is notlimited to, an antibody a tetrameric immunoglobulin, an IgG molecule, anIgG₁ molecule, a monoclonal antibody, a chimeric antibody, a CDR-graftedantibody, a humanized antibody, an affinity matured antibody, andfragments of any such antibodies that retain the ability to bind to anantigen.

“Capture molecule” as used herein refers to a specific binding partneror specific binding member used to capture or immobilize an analyte ofinterest in a biological sample. A capture molecule is often onecomponent of a complex in addition to the analyte of interest and mayalso contain one or more detection molecules. The complex may optionallybe bound to a solid support.

“Component,” “components,” or “at least one component,” refer generallyto a capture antibody, a detection reagent or conjugate, a calibrator, acontrol, a sensitivity panel, a container, a buffer, a diluent, a salt,an enzyme, a co-factor for an enzyme, a detection reagent, apretreatment reagent/solution, a substrate (e.g., as a solution), a stopsolution, and the like that can be included in a kit for assay of a testsample, such as patient urine, serum, whole blood, tissue aspirate, orplasma sample, in accordance with the methods described herein and othermethods known in the art. Some components can be in solution orlyophilized for reconstitution for use in an assay.

“Contacting” and grammatical equivalents thereof as used herein refer toany type of combining action which brings a binding member intosufficiently close proximity with the analyte of interest in the samplesuch that a binding interaction will occur if the analyte of interestspecific for the binding member is present in the sample. Contacting maybe achieved in a variety of different ways, including combining thesample with a binding member, exposing a target analyte to a bindingmember by introducing the binding member in close proximity to theanalyte, and the like.

“Control” as used herein refers to a reference standard for an analytesuch as is known or accepted in the art, or determined empirically usingacceptable means such as are commonly employed. A “reference standard”is a standardized substance which is used as a measurement base for asimilar substance. For example, there are documented reference standardspublished in the U.S. Pharmacopeial Convention (USP-NF), Food ChemicalsCodex, and Dietary Supplements Compendium (all of which are available athttp://www.usp.org), and other well-known sources. Methods forstandardizing references are described in the literature. Alsowell-known are means for quantifying the amounts of analyte present byuse of a calibration curve for analyte or by comparison to an alternatereference standard. A standard curve can be generated using serialdilutions or solutions of known concentrations of analyte, by massspectroscopy, gravimetric methods, and by other techniques known in theart. Alternate reference standards that have been described in theliterature include standard addition (also known as the method ofstandard addition), or digital polymerase chain reaction.

“Detection molecule” as used herein refers to a specific binding partneror specific binding member that is used to detect the presence of and/orquantify or measure the amount of an analyte of interest in a biologicalsample. A detection molecule is often one component of a complex thatmay contain a one or more capture molecules and an analyte of interest.The complex may optionally be bound to a solid support.

“Immobilized” as used herein, refers to a stable association of thefirst specific binding member with a surface of a solid support. By“stable association” is meant a physical association between twoentities in which the mean half-life of association is one day or more,e.g., under physiological conditions. In certain aspects, the physicalassociation between the two entities has a mean half-life of two days ormore, one week or more, one month or more, including six months or more,e.g., 1 year or more, in PBS at 4° C. According to certain embodiments,the stable association arises from a covalent bond between the twoentities, a non-covalent bond between the two entities (e.g., an ionicor metallic bond), or other forms of chemical attraction, such ashydrogen bonding, Van der Waals forces, and the like.

“Label” and “detectable label” as used herein refer to a moiety attachedto an antibody or an analyte to render the reaction between the antibodyand the analyte detectable, and the antibody or analyte so labeled isreferred to as “detectably labeled.” A label can produce a signal thatis detectable by visual or instrumental means. Various labels includesignal-producing substances, such as chromogens, fluorescent compounds,chemiluminescent compounds, radioactive compounds, and the like. Otherlabels are described herein. In this regard, the moiety, itself, may notbe detectable but may become detectable upon reaction with yet anothermoiety. Use of the term “detectably labeled” is intended to encompasssuch labeling.

“Monoclonal antibody” as used herein refers to an antibody obtained froma population of substantially homogeneous antibodies, i.e., theindividual antibodies comprising the population are identical except forpossible naturally occurring mutations that may be present in minoramounts. Monoclonal antibodies are highly specific, being directedagainst a single antigen. Furthermore, in contrast to polyclonalantibody preparations that typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen. Themonoclonal antibodies herein specifically include “chimeric” antibodiesin which a portion of the heavy and/or light chain is identical with orhomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is identical with orhomologous to corresponding sequences in antibodies derived from anotherspecies or belonging to another antibody class or subclass, as well asfragments of such antibodies, so long as they exhibit the desiredbiological.

“Polynucleotides” or “oligonucleotides” refer to nucleobase polymers oroligomers in which the nucleobases are connected by sugar phosphatelinkages (sugar-phosphate backbone). Exemplary poly- andoligonucleotides include polymers of 2′-deoxyribonucleotides (DNA) andpolymers of ribonucleotides (RNA). A polynucleotide may be composedentirely of ribonucleotides, entirely of 2′-deoxyribonucleotides orcombinations thereof. “Nucleic acid” encompasses “polynucleotide” and“oligonucleotides” and includes single stranded and double strandedpolymers of nucleotide monomers.

“Predetermined cutoff” and “predetermined level” as used herein refer toan assay cutoff value that is used to assess diagnostic, prognostic, ortherapeutic efficacy results by comparing the assay results against thepredetermined cutoff/level, where the predetermined cutoff/level alreadyhas been linked or associated with various clinical parameters (e.g.,presence of disease, stage of disease, severity of disease, progression,non-progression, or improvement of disease, etc.). The disclosureprovides exemplary predetermined levels. However, it is well-known thatcutoff values may vary depending on the nature of the immunoassay (e.g.,antibodies employed, reaction conditions, sample purity, etc.). Itfurther is well within the ordinary skill of one in the art to adapt thedisclosure herein for other immunoassays to obtain immunoassay-specificcutoff values for those other immunoassays based on the descriptionprovided by this disclosure. Whereas the precise value of thepredetermined cutoff/level may vary between assays, the correlations asdescribed herein should be generally applicable.

“Pretreatment reagent,” e.g., lysis, precipitation and/or solubilizationreagent, as used in a diagnostic assay as described herein is one thatlyses any cells and/or solubilizes any analyte that is/are present in atest sample. Pretreatment is not necessary for all samples, as describedfurther herein. Among other things, solubilizing the analyte entailsrelease of the analyte from any endogenous binding proteins present inthe sample. A pretreatment reagent may be homogeneous (not requiring aseparation step) or heterogeneous (requiring a separation step). Withuse of a heterogeneous pretreatment reagent, there is removal of anyprecipitated analyte binding proteins from the test sample prior toproceeding to the next step of the assay. The pretreatment reagentoptionally can comprise: (a) one or more solvents and salt, (b) one ormore solvents, salt and detergent, (c) detergent, (d) detergent andsalt, or (e) any reagent or combination of reagents appropriate for celllysis and/or solubilization of analyte.

“Quality control reagents” in the context of immunoassays and kitsdescribed herein, include, but are not limited to, calibrators,controls, and sensitivity panels. A “calibrator” or “standard” typicallyis used (e.g., one or more, such as a plurality) in order to establishcalibration (standard) curves for interpolation of the concentration ofan analyte, such as an antibody or an analyte. Alternatively, a singlecalibrator, which is near a predetermined positive/negative cutoff, canbe used. Multiple calibrators (i.e., more than one calibrator or avarying amount of calibrator(s)) can be used in conjunction to comprisea “sensitivity panel.”

“Receptor” as used herein refers to a protein-molecule that recognizesand responds to endogenous-chemical signals. When suchendogenous-chemical signals bind to a receptor, they cause some form ofcellular/tissue-response. Examples of receptors include, but are notlimited to, neural receptors, hormonal receptors, nutrient receptors,and cell surface receptors.

“Recombinant antibody” and “recombinant antibodies” refer to antibodiesprepared by one or more steps, including cloning nucleic acid sequencesencoding all or a part of one or more monoclonal antibodies into anappropriate expression vector by recombinant techniques and subsequentlyexpressing the antibody in an appropriate host cell. The terms include,but are not limited to, recombinantly produced monoclonal antibodies,chimeric antibodies, humanized antibodies (fully or partiallyhumanized), multi-specific or multi-valent structures formed fromantibody fragments, bifunctional antibodies, heteroconjugate Abs,DVD-Ig®s, and other antibodies as described in (i) herein.(Dual-variable domain immunoglobulins and methods for making them aredescribed in Wu, C., et al., Nature Biotechnology, 25:1290-1297 (2007)).The term “bifunctional antibody,” as used herein, refers to an antibodythat comprises a first arm having a specificity for one antigenic siteand a second arm having a specificity for a different antigenic site,i.e., the bifunctional antibodies have a dual specificity.

“Sample,” “test sample,” “biological sample,” “sample from a subject,”“fluid biological sample,” and “patient sample” as used herein may beused interchangeable and refer to fluid sample containing or suspectedof containing an analyte of interest.

As used herein, “signal generating compound” refers to any molecule,compound, protein or the like that can be converted to a detectableproduct or detectable label upon exposure to a suitable or appropriateconverting agent, such as a signal generating substrate. A “detectableproduct” or “detectable label” is any molecule, particle, or the like,that facilitates detection, by acting as the detected entity, using achosen technique known in the art. An example of a signal generatingcompound is an enzyme such as amylases, polynucleotidase, arginase,adenase, aminopolypeptidase, pepsin, lipases, catalase, tyrosinases,alcohol dehydrogenase, succinic dehydrogenase, diaphorase, glyoxalase,aldolase, glucose oxidase, horseradish peroxidase, a galactosidase (suchas beta-galactosidase), phosphatases, phosphorylases and hexokinases orcombinations thereof.

As used herein “signal generating substrate” refers any molecule,compound, protein, substance, particle, or the like, that can beconverted to or result in a signal generating compound being convertedto a detectable product or detectable label upon exposure to a suitableor appropriate converting agent, such as a signal generating compound. A“detectable compound” or “detectable label” is any molecule, particle,or the like, that facilitates detection, by acting as the detectedentity, using a chosen technique known in the art. Signal generatingsubstrates can be colorimetric, chemiluminescent or chemifluorescent. Anexample of a signal generating substrate is an enzymatic substrate, suchas a chemiluminescent substrate such as CDP-Star®, (disodium4-chloro-3-(methoxyspiro{1,2-dioxetane-3,2′-(5′-chloro)tricyclo[3.3.1.1.s-up.3,7]decane}-4-yl)phenylphosphate), CSPD®, or (disodium3-(4-methoxyspiro{1,2-dioxetane-3,2-(5′-chloro)tricyclo[3.3.1.1-.sup.3,7]decane}-4-yl)phenylphosphate); a luminescent substrate such as p-nitrophenyl phosphate,5-bromo-4-chloro-3-indolyl phosphate (BCIP), 4-nitro blue tetrazoliumchloride (NBT), or iodonitrotetrazolium (INT); a fluorescent substratesuch as 4-methylumbelliferyl phosphate (4-MUP); and a chromogenicsubstrate such as 5-bromo-4-chloro-3-indolyl phosphate (BCIP), disodium5-bromo-6-chloro-indolyl phosphate, or p-nitrophenyl phosphate.

“Specific binding partner” or “specific binding member” as usedinterchangeably herein refers to one of two or more different moleculesthat specifically recognize the other molecule compared to substantiallyless recognition of other molecules. The one of two different moleculeshas an area on the surface or in a cavity, which specifically binds toand is thereby defined as complementary with a particular spatial andpolar organization of the other molecule. The molecules may be membersof a specific binding pair. For example, a specific binding member mayinclude, but is not limited to, a protein, such as a receptor, anenzyme, and an antibody.

In addition to antigen and antibody specific binding pairs of commonimmunoassays, other specific binding pairs can include biotin and avidin(or streptavidin), carbohydrates and lectins, complementary nucleotidesequences, effector and receptor molecules, cofactors and enzymes,enzymes and enzyme inhibitors, and the like. Furthermore, specificbinding pairs can include members that are analogs of the originalspecific binding members, for example, an analyte-analog. Immunoreactivespecific binding members include antigens, antigen fragments, andantibodies, including monoclonal and polyclonal antibodies as well ascomplexes and fragments thereof, whether isolated or recombinantlyproduced.

“Solid phase” or “solid support” as used interchangeably herein, refersto any material that is insoluble, or can be made insoluble by asubsequent reaction. The solid phase can be chosen for its intrinsicability to attract and immobilize a capture agent. Alternatively, thesolid phase can have affixed thereto a linking agent that has theability to attract and immobilize the capture agent. For example, thelinking agent can include a charged substance that is oppositely chargedwith respect to the capture agent itself or to a charged substanceconjugated to the capture agent. In general, the linking agent can beany binding partner (preferably specific) that is immobilized on(attached to) the solid phase and that has the ability to immobilize thecapture agent through a binding reaction. The linking agent enables theindirect binding of the capture agent to a solid phase material beforethe performance of the assay or during the performance of the assay. Forexamples, the solid phase can be plastic, derivatized plastic, magnetic,or non-magnetic metal, glass or silicon, including, for example, a testtube, microtiter well, sheet, bead, microparticle, chip, and otherconfigurations known to those of ordinary skill in the art.

“Subject” and “patient” as used herein interchangeably refers to anyvertebrate, including, but not limited to, a mammal (e.g., cow, pig,camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat,dog, rat, and mouse, a non-human primate (for example, a monkey, such asa cynomolgous or rhesus monkey, chimpanzee, etc.) and a human). In someembodiments, the subject may be a human or a non-human. The subject orpatient may be undergoing other forms of treatment.

“Threshold” as used herein refers to an empirically determined andsubjective cutoff level above which acquired data is considered“signal,” and below which acquired data is considered “noise.” Acomputer program based on CUSUM (Cumulative Sums Algorithm) is employedto process acquired data and detect events based on threshold input fromthe user. Variation between users is avoided by detection of any manyevents as possible followed by filtering the data afterwards forspecific purposes. With a “loose” threshold a lesser number of eventswill be counted as signal. With a “tight” threshold a greater number ofevents will be counted as signal. Setting the threshold as loose ortight is a subjective choice based on the desired sensitivity orspecificity for an assay, and whether in a given assessment falsepositives or false negatives would be preferred.

“Treat”, “treating” or “treatment” are each used interchangeably hereinto describe reversing, alleviating, or inhibiting the progress of adisease, or one or more symptoms of such disease, to which such termapplies. Depending on the condition of the subject, the term also refersto preventing a disease, and includes preventing the onset of a disease,or preventing the symptoms associated with a disease. A treatment may beeither performed in an acute or chronic way. The term also refers toreducing the severity of a disease or symptoms associated with suchdisease prior to affliction with the disease. Such prevention orreduction of the severity of a disease prior to affliction refers toadministration of an antibody or pharmaceutical composition of thepresent invention to a subject that is not at the time of administrationafflicted with the disease. “Preventing” also refers to preventing therecurrence of a disease or of one or more symptoms associated with suchdisease. “Treatment” and “therapeutically,” refer to the act oftreating, as “treating” is defined above.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. In case of conflict, the present document, includingdefinitions, will control. Preferred methods and materials are describedbelow, although methods and materials similar or equivalent to thosedescribed herein can be used in practice or testing of the presentinvention. All publications, patent applications, patents and otherreferences mentioned herein are incorporated by reference in theirentirety to disclose and describe the methods and/or materials inconnection with which the publications are cited. The materials,methods, and examples disclosed herein are illustrative only and notintended to be limiting.

2. Improved Signal-Generating Digital Assays

The present invention relates to improved assays, such as fluorescentimmunoassays, that employ an array of reaction vessels (such as one ormore wells), are sealed using solvent well sealing, and exhibit reducedbackground noise.

In fluorescent immunoassays, a single molecule of signal generatingcompound can generate a detectable signal by accumulating the detectablelabel produced from the signal generating substrate in a femto-literchamber. While digital ELISAs have vast potential, one of the drawbacksis that the assay requires a number of steps to be performed. Thesesteps include: 1) forming a capture molecule-analyte ofinterest-detection molecule complex on a solid support; 2) entrappingthe complex in a droplet; 3) moving the droplet into a reaction vesseland adding a signal generation substrate (either simultaneously orsequentially with the droplet in any order) to form an aqueous phase, 4)adding a solvent, such as fluorinated oil (FC40), to the reactionvessel, causing the aqueous and solvent phases to switch therebyresulting in the separation of the aqueous solution in the reactionvessel; 5) removing the aqueous phase (which is the upper or top layerin the reaction vessel); and 6) acquiring an image of the reactionvessel using a digital counting device (such as an optical microscope).The removal of the aqueous phase in step 5 is for the purposes ofeliminating background signal phase but is very time consuming.Specifically, one skilled in the art has to make sure to remove theaqueous phase completely because incomplete removal of the aqueous phasewill result in residual background signal (fluorescence or glow noise).This background noise is caused by signal (fluorescence or glow) presentin the upper aqueous phase and obstructs the counting of the number ofsignal generating droplets under the digital counting device (such as anoptical microscope).

To delete the signal background noise, the present invention involves animproved method of conducting a digital assay that involves adding oneor more colorants, such as a dye component (e.g. black ink), into thesolution phase (aqueous or sealant (e.g., oil)) before or after addingthe droplet or before or after adding the droplet and the signalgenerating substrate to the reaction vessel. As a result, the additionof the colorant (such as black ink or mixed black ink) obtains the sameresult as removing the aqueous phase, thus eliminating the need for thisstep in the assay completely. In the dye mixing method described herein,the colorant suppresses not only the background signal noise in theaqueous phase which is separated by the solvent addition, but alsosuppresses the background signal noise anywhere in the digital countingdevice. As a result, true signals, free from background, can be easilyobtained from the image with the deepest shade of color (such as black).While not wishing to be bound by any theory, it is believed that thereason that such true signals can be obtained is that the light pathlength of the reaction vessel is very small (for example, if afemto-liter reaction vessel is used, the chamber is only 4 micrometers),and the transmissivity of the true signal is not affected or beinterfered by the colorant in the solution.

The present disclosure describes improved methods for measuring ordetecting an analyte present in a fluid biological sample using an arraycomprising a plurality of reaction vessels using the solvent sealingmethod which involves a sealant. For example, FIGS. 1 and 2 showschematics of a digital immunoassay using a drip oil sealing method. The‘digital ELISA’ is based on the array device of a million of femto-litersize of water-in oil (W/O) droplets. The digital immunoassay may be anultra-high sensitive immunoassay, such as a digital ELISA that coulddetect target molecules around the concentration of atto to sub-femtomol/L level (atto=10⁻¹⁸, femto=10⁻¹⁵). Other methods of drip oil sealinginclude those described in, for example, Rondelez et al., Naturebiotechnology 23(3):361-365 (2005), Kim et al., Lab on a Chip12(23):4986-4991(2012), Japan Patent Number 3727026, and InternationalPatent Publication Numbers WO2012/121310 and WO2016/006208, the contentsof each of which are herein incorporated by reference. In someembodiments, the signal-generating digital immunoassay includes afemto-liter droplet array.

The method for measuring or detecting the analyte includes exposing orcontacting the fluid biological sample to a plurality of solid supportsto create a mixture, each solid support comprises at least one firstspecific binding member (“binding members” alternately referred to as“specific binding members,” and as described below) capable of bindingto the analyte, wherein at least some fraction of the specific bindingmembers bind to the analyte thereby producing solid support-firstspecific binding member-analyte complexes and at least some fraction ofthe specific binding members does not bind to any analyte; adding to orcontacting with the mixture one or more second specific binding memberscapable of binding to the analyte thereby producing solid support-firstspecific binding member-analyte-second specific binding membercomplexes, the second specific binding member comprising a signalgenerating compound; adding a signal generating substrate to the solidsupport-first specific binding member-analyte-second specific bindingmember complexes; and spatially segregating at least a portion of thesolid support-first specific binding member-analyte-second specificbinding member complexes into a plurality of separate locations. In theimproved method, a colorant is added before or after spatiallysegregating at least a portion of the solid support-first specificbinding member-analyte-second specific binding member complexes into aplurality of separate locations (i.e., femto-liter chamber formation).

The plurality of reaction vessels are covered with a sealant, such as aheavy fluorinated oil, and the aqueous phase and the sealant phase(e.g., oil phase) are changed. In some embodiments, the aqueous phaseand the sealant phase (e.g., oil phase) are changed by tilting theplurality of reaction vessels. In some embodiments, the heavy fluorinateoil is FC-40, FC-72, FC-84, FC-77, FC-3255, FC-3283, FC-43, FC-70), 3MNovec 4200, 3M Novec 4300, 3M FC-4432, 3M FC-4430, or 3M FC-4434. Afterthe aqueous phase and the sealant phase (e.g., oil phase) are changed,the aqueous phase is removed. The plurality of reaction vessels aremoved to a digital counting device, such as a fluorescence microscopeand the presence or absence of the detectable signal is detected. Thedetection of the presence of the detectable signal indicates thepresence of a single molecule of analyte in the sample. In someembodiments, the detectable signal is detected using an opticalmicroscope, such as a fluorescence microscope to acquire fluorescenceimages. In some embodiments, the detectable signal is detected using afluorescence microscope to acquire fluorescence images.

In some embodiments, the plurality of reaction vessels can be amicrowell array or nanowell array. In some embodiments, the microwellarray or nanowell array has a diameter of at least about 4 mm, at leastabout 5 mm, at least about 6 mm, at least about 7 mm, at least about 8mm, at least about 9 mm, or at least about 10 mm. In some embodiments,the microwell array has a diameter of 6 mm. In some embodiments, themicrowell array or nanowell array contains approximately 100,000 toapproximately 1,000,000 wells, approximately 200,000 to approximately750,000 wells, or approximately 300,000 to approximately 500,000 wells.In some embodiments, the microwell array contains about 100,000, about200,000, about 300,000, about 350,000, about 375,000, about 400,000,about 425,000, about 450,000, about 475,000, about 500,000, about600,000, about 700,000, about 800,000, about 900,000, or about 1,000,000wells. In some embodiments, the microwell array contains 400,000 wells.In some embodiments, the wells can have at least about 1 μM diameter, atleast about 2 μM diameter, at least about 3 μM diameter, at least about4 μM diameter, at least about 5 μM diameter, at least about 6 μMdiameter, at least about 7 μM diameter, at least about 8 μM diameter, atleast about 9 μM diameter, or at least about 10 μM diameter at thebottom of the well. In some embodiments, the plurality of reactionvessels can be a microwell array or nanowell array having a diameter of6 mm and containing approximately 400,000 wells having a 5 μm diameterat the bottom of the well.

Following complex formation between the immobilized first specificbinding member and the analyte, any unbound analyte may be removed fromthe vicinity of the first specific binding member along with the samplewhile the complex of the first specific binding member and the analytemay be retained due to its association with the solid support.Optionally, the solid support may be contacted with a wash buffer toremove any molecules non-specifically bound to the solid support.

After the first contacting step, and the optional removal of sampleand/or optional wash steps, the complex of the first specific bindingmember and the analyte may be contacted with a second specific bindingmember, thereby leading to the formation of a sandwich complex in whichthe analyte is bound by the two binding members. An optional mixing ofthe second member with the first specific binding member-analyte complexmay be carried out during the second contacting step. In someembodiments, immobilization of the analyte molecules with respect to asurface may aid in removal of any excess second specific binding membersfrom the solution without concern of dislodging the analyte moleculefrom the surface. In some embodiments, the second specific bindingmember may include signal generating compound, attached thereto.

As noted above, the second contacting step may be carried out inconditions sufficient for binding interaction between the analyte andthe second specific binding member. Following the second contactingstep, any unbound second specific binding member may be removed,followed by an optional wash step. In some embodiments, the secondspecific binding member not bound to the analyte bound to the firstspecific binding member is removed before spatially segregating at leasta portion of the solid support-first specific bindingmember-analyte-second specific binding member complexes into a pluralityof separate locations.

In some embodiments, the signal generating compound can be alkalinephosphatase or β-galactosidase (beta-Gal). In some embodiments, thesignal generating substrate can be a fluorescent substrate for thesignal generating compound. For example, the signal generating substratecan be 4-methylumbelliferyl phosphate (MUP), fluorescein diphosphate(FDP), 6,8-Difluoro-4-Methylumbelliferyl Phosphate (DiFMUP), or9H-(1,3-Dichloro-9,9-Dimethylacridin-2-One-7-yl) Phosphate (DDAOPhosphate). In some embodiments, the method further includes aninhibitor of the signal generating compound in the reaction mixture. Insome embodiments, the inhibitor is levamisole.

In some embodiments, the method further includes incubating the mixturefor a period of time before adding to the mixture one or more secondspecific binding members or after adding to the mixture one or moresecond specific binding members, wherein the period of time is anincubation period and is about 40 minute to about 300 minutes. In someembodiments, the fluid sample is serum, plasma or a whole blood sample.

In some embodiments, the method further includes contacting the fluidsample with one or more detergents, a surfactant, a nonpolar solvent,sonication, heating, or combination thereof, prior to contacting thefluid sample to the plurality of solid supports. In some embodiments,the method further includes the step of calculating a cut-off valueusing the formula: CO=2S/N, S/N CO=2 S/N, S/N ratio was calculated %signal from serum specimen with analyte divided by % signal from serumspecimen without analyte. In some embodiments, the solid support is amagnetic solid support.

The present disclosure describes methods of determining the presence orabsence of a single molecule of an analyte in a fluid sample. The methodincludes contacting the fluid sample containing or suspected ofcontaining the analyte with a plurality of solid supports to create amixture. Each solid support includes at least one first specific bindingmember capable of binding to the analyte thereby producing solidsupport-first specific binding member-analyte complexes. The solidsupport-first specific binding member-analyte complexes in the mixtureare washed with a first wash buffer to remove any solid support-firstspecific binding member not bound to the analyte thereby producing awashed mixture. The one or more second specific binding members capableof binding to the analyte are added to the washed mixture therebyproducing solid support-first specific binding member-analyte-secondspecific binding member complexes. The second specific binding membercomprises a signal generating compound attached thereto. The solidsupport-first specific binding member-analyte-second specific bindingmember complexes are washed with a second wash buffer to remove anysecond specific binding member not bound to the analyte bound to thefirst specific binding member thereby producing washed solid supportcomplexes. A signal generating substrate is added to the washed solidsupport complexes. The signal generating compound and the signalgenerating substrate produce a detectable signal. At least a portion ofthe washed solid support complexes are spatially segregating into aplurality of separate location. The plurality of separation locationsincludes a microwell array, as described above. The microwells ornanowells are covered with a sealant, e.g., heavy fluorinated oil. Theaqueous phase and the sealant phase (e.g., oil phase) are changed. Thepresence or absence of the detectable signal is detected using a digitalcounting device. The detection of the presence of the detectable signalindicates the presence of a single molecule of the analyte in thesample. In the improved method, a colorant is added before or afterspatially segregating the washed solid support complexes into aplurality of separate locations (i.e., femto-liter chamber formation).

In some embodiments, the sensitivity of detection of the analyte is atleast about 0.1 fM to at least about 10 fM, at least about 0.5 mIU/mL,or at least about 0.24 pg/mL. In some embodiments, the sensitivity ofdetection of the analyte is at least about 0.05 fM, at least about 0.06fM, at least about 0.07 fM, at least about 0.08 fM, at least about 0.09fM, at least about 0.10 fM, at least about 0.11 fM, at least about 0.12fM, at least about 0.13 fM, at least about 0.14 fM, at least about 0.15fM, at least about 0.5 fM, at least about 1.0, at least about 5 fM, atleast about 10 fM, at least about 20 fM, at least about 30 fM, at leastabout 40 fM, at least about 50 fM, at least about 60 fM, at least about70 fM, at least about 80 fM, at least about 90 fM, or at least about 100fM.

For example, the disclosed methods may be used for measuring ordetecting an analyte present in a biological sample or for diagnosing apatient or screening a blood supply.

In exemplary cases, the method may include contacting the sample with afirst specific binding member (“binding members” alternately referred toas “specific binding members,” and as described below), where the firstspecific binding member is immobilized on a solid support and where thefirst specific binding member specifically binds to the analyte;contacting the analyte with a second specific binding member, whichsecond specific binding member specifically binds to the analyte andwhich second specific binding member includes a signal generatingcompound or signal generating substrate described below.

In certain cases, the first specific binding member may be immobilizedon a solid support. The solid support having a surface on which thebinding reagent (such as one or more specific binding members) isimmobilized may be any convenient surface in planar or non-planarconformation, such as a surface of a microfluidic chip, an interiorsurface of a chamber, an exterior surface of a bead (as defined herein),or an interior and/or exterior surface of a porous bead. For example,the first specific binding member may be attached covalently ornon-covalently to a bead, e.g., latex, agarose, sepharose, streptavidin,tosylactivated, epoxy, polystyrene, amino bead, amine bead, carboxylbead, or the like. In certain embodiments, the bead may be a particle,e.g., a microparticle. In some embodiments, the microparticle may bebetween about 0.1 nm and about 10 microns, between about 50 nm and about5 microns, between about 100 nm and about 1 micron, between about 0.1 nmand about 700 nm, between about 500 nm and about 10 microns, betweenabout 500 nm and about 5 microns, between about 500 nm and about 3microns, between about 100 nm and 700 nm, or between about 500 nm and700 nm. For example, the microparticle may be about 4-6 microns, about2-3 microns, or about 0.5-1.5 microns. Particles less than about 500 nmare sometimes considered nanoparticles. Thus, the microparticleoptionally may be a nanoparticle between about 0.1 nm and about 500 nm,between about 10 nm and about 500 nm, between about 50 nm and about 500nm, between about 100 nm and about 500 nm, about 100 nm, about 150 nm,about 200 nm, about 250 nm, about 300 nm, about 350 nm, about 400 nm,about 450 nm, or about 500 nm.

In certain embodiments, the bead may be a magnetic bead or a magneticparticle. Magnetic beads/particles may be ferromagnetic, ferrimagnetic,paramagnetic, superparamagnetic or ferrofluidic. Exemplary ferromagneticmaterials include Fe, Co, Ni, Gd, Dy, CrO₂, MnAs, MnBi, EuO, NiO/Fe.Examples of ferrimagnetic materials include NiFe₂O₄, CoFe₂O₄, Fe₃O₄ (orFeO.Fe₂O₃). Beads can have a solid core portion that is magnetic and issurrounded by one or more non-magnetic layers. Alternately, the magneticportion can be a layer around a non-magnetic core. The solid support onwhich the first specific binding member is immobilized may be stored indry form or in a liquid. The magnetic beads may be subjected to amagnetic field prior to or after contacting with the sample with amagnetic bead on which the first specific binding member is immobilized.

After the contacting step, the sample and the first specific bindingmember may be incubated for a sufficient period of time to allow for thebinding interaction between the binding member and analyte to occur. Inaddition, the incubating may be in a binding buffer that facilitates thespecific binding interaction. The binding affinity and/or specificity ofthe first specific binding member and/or the second specific bindingmember may be manipulated or altered in the assay by varying the bindingbuffer. In some embodiments, the binding affinity and/or specificity maybe increased by varying the binding buffer. In some embodiments, thebinding affinity and/or specificity may be decreased by varying thebinding buffer.

The binding affinity and/or specificity of the first specific bindingmember and/or the second specific binding member may be measured usingthe disclosed methods described below. In some embodiments, the onealiquot of sample is assayed using one set of conditions and compared toanother aliquot of sample assayed using a different set of conditions,thereby determining the effect of the conditions on the binding affinityand/or specificity. For instance, changing or altering the condition canbe one or more of removing the target analyte from the sample, adding amolecule that competes with the target analyte or the ligand forbinding, and changing the pH, salt concentration, or temperature.Additionally or alternatively, a duration of time can be the variableand changing the condition may include waiting for a duration of timebefore again performing the detection methods.

The binding buffer may include molecules standard for antigen-antibodybinding buffers such as, albumin (e.g., BSA), non-ionic detergents(Tween-20, Triton X-100), and/or protease inhibitors (e.g., PMSF). Incertain cases, the binding buffer may be added to the microfluidic chip,chamber, etc., prior to or after adding the sample. In certain cases,the first specific binding member may be present in a binding bufferprior to contacting with the sample. The length of time for bindinginteraction between the binding member and analyte to occur may bedetermined empirically and may depend on the binding affinity andbinding avidity between the binding member and the analyte. In certainembodiments, the contacting or incubating may be for a period of 5 secto 1 hour, such as, 10 seconds to 30 minutes, or 1 minute to 15 minutes,or 5 minutes to 10 minutes, e.g., 10 seconds, 15 seconds, 30 seconds, 1minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1hour or 2 hours. Other conditions for the binding interaction, such as,temperature, salt concentration, may also be determined empirically ormay be based on manufacturer's instructions. For example, the contactingmay be carried out at room temperature (21° C.-28° C., e.g., 23° C.-25°C.), 37° C., or 4° C. In certain embodiments, an optional mixing of thesample with the first specific binding member may be carried out duringthe contacting step.

Following complex formation between the immobilized first specificbinding member and the analyte, any unbound analyte may be removed fromthe vicinity of the first specific binding member along with the samplewhile the complex of the first specific binding member and the analytemay be retained due to its association with the solid support.Optionally, the solid support may be contacted with a wash buffer toremove any molecules non-specifically bound to the solid support.

After the first contacting step, and the optional removal of sampleand/or optional wash steps, the complex of the first specific bindingmember and the analyte may be contacted with a second specific bindingmember, thereby leading to the formation of a sandwich complex in whichthe analyte is bound by the two binding members. An optional mixing ofthe second member with the first specific binding member-analyte complexmay be carried out during the second contacting step. In someembodiments, immobilization of the analyte molecules with respect to asurface may aid in removal of any excess second specific binding membersfrom the solution without concern of dislodging the analyte moleculefrom the surface. In some embodiments, the second specific bindingmember may include a signal generating compound, attached thereto.

As noted above, the second contacting step may be carried out inconditions sufficient for binding interaction between the analyte andthe second specific binding member. Following the second contactingstep, any unbound second specific binding member may be removed,followed by an optional wash step. Any unbound second specific bindingmember may be separated from the complex of the first specific bindingmember-analyte-second specific binding member by a suitable means suchas, droplet actuation, electrophoresis, electrowetting,dielectrophoresis, electrostatic actuation, electric field mediated,electrode mediated, capillary force, chromatography, centrifugation, oraspiration. Upon removal of any unbound second specific binding memberfrom the vicinity of the complex of the first specific bindingmember-analyte-second specific binding member, the signal generatingcompound attached to the second specific binding member present in thecomplex of the first specific binding member-analyte-second specificbinding member may be separated by a suitable means.

In certain embodiments, the separation of the signal generating compoundfrom the first specific binding member-analyte-second specific bindingmember complex is carried out under conditions that do not result indisruption of the complex, resulting in release of only the signalgenerating compound from the complex. In other cases, the separation ofthe signal generating compound from the first specific bindingmember-analyte-second specific binding member complex is carried outunder conditions that may result in disruption of the complex, resultingin release of the signal generating compound, as well as one or more ofthe second specific binding member, the analyte, the first specificbinding member from the complex.

The number of signal generating compound molecules can be correlated tothe number of analyte molecules in the complex which are proportional tothe concentration of the analyte in the sample. In certain embodiments,the correlation between the signal generating compound and the analyteconcentration may be direct (higher number of signal generating compoundmolecules relates to higher analyte concentration). In embodiments wherea signal generating compound-tagged competitor or analyte, such as atracer (as defined herein), is combined with the sample, which signalgenerating compound-tagged competitor or analyte competes with theanalyte in the sample for binding to the first specific binding member,the correlation between the signal generating compound and the analyteconcentration may be inverse (lower number of signal generating compoundmolecules relates to higher analyte concentration). The correlationbetween the number of signal generating compound molecules and analyteconcentration, whether direct or inverse, may be linear or logarithmic.

In certain embodiments, the simultaneous analysis of multiple analytesin a single sample may be performed by using a plurality of differentfirst and second specific binding members where a pair of first andsecond specific binding members is specific to a single analyte in thesample. In these embodiments, the signal generating compound associatedwith the second specific binding member of a first pair of first andsecond specific binding members specific to a single analyte may bedistinguishable from the signal generating compound or signal generatingsubstrate associated with the second specific binding member of a secondpair of first and second specific binding members specific to adifferent analyte. As noted above, a first signal generating compoundmay be distinguishable from second signal generating compound or signalgenerating substrate based on difference in substrates.

In some embodiments, the concentration of an analyte in the fluid samplethat may be substantially accurately determined is less than about 5000fM (femtomolar), less than about 3000 fM, less than about 2000 fM, lessthan about 1000 fM, less than about 500 fM, less than about 300 fM, lessthan about 200 fM, less than about 100 fM, less than about 50 fM, lessthan about 25 fM, less than about 10 fM, less than about 5 fM, less thanabout 2 fM, less than about 1 fM, less than about 500 aM (attomolar),less than about 100 aM, less than about 10 aM, less than about 5 aM,less than about 1 aM, less than about 0.1 aM, less than about 500 zM(zeptomolar), less than about 100 zM, less than about 10 zM, less thanabout 5 zM, less than about 1 zM, less than about 0.1 zM, or less.

In some cases, the limit of detection (e.g., the lowest concentration ofan analyte which may be determined in solution) is about 100 fM, about50 fM, about 25 fM, about 10 fM, about 5 fM, about 2 fM, about 1 fM,about 500 aM (attomolar), about 100 aM, about 50 aM, about 10 aM, about5 aM, about 1 aM, about 0.1 aM, about 500 zM (zeptomolar), about 100 zM,about 50 zM, about 10 zM, about 5 zM, about 1 zM, about 0.1 zM, or less.In some embodiments, the concentration of analyte in the fluid samplethat may be substantially accurately determined is between about 5000 fMand about 0.1 fM, between about 3000 fM and about 0.1 fM, between about1000 fM and about 0.1 fM, between about 1000 fM and about 0.1 zM,between about 100 fM and about 1 zM, between about 100 aM and about 0.1zM, or less.

The upper limit of detection (e.g., the upper concentration of ananalyte which may be determined in solution) is at least about 100 fM,at least about 1000 fM, at least about 10 pM (picomolar), at least about100 pM, at least about 100 pM, at least about 10 nM (nanomolar), atleast about 100 nM, at least about 1000 nM, at least about 10 μM, atleast about 100 μM, at least about 1000 μM, at least about 10 mM, atleast about 100 mM, at least about 1000 mM, or greater.

In some cases, the presence and/or concentration of the analyte in asample may be detected rapidly, usually in less than about 1 hour, e.g.,45 minutes, 30 minutes, 15 minutes, 10 minutes, 5 minutes, 1 minute, or30 seconds.

In some embodiments, one or more detected signals correspond to abinding event of a binding member to an analyte. In some embodiments,one detected signal corresponds to a binding event of a binding memberto an analyte. In some embodiments, two or more detected signalscorrespond to a binding event of a binding member to an analyte.

In some embodiments, the solid support comprising the first specificbinding member and second specific binding member are added sequentiallyor simultaneously to the sample.

a) Colorant

In the present invention, a colorant is added before or afterfemto-liter chamber formation. The colorant suppresses the glow of thefluorescent substance in the upper phase without the need for the timeconsuming step of removing the aqueous phase. In some embodiments, thecolorant is added simultaneously with the signal generating substrate.In some embodiments, the colorant is added to the washed solid supportcomplexes before the signal generating substrate. In some embodiments,the colorant is added to the washed solid support complexes after thesignal generating substrate. In some embodiments, the colorant is addedto the displaced aqueous phase or to the sealant phase (e.g., oilphase).

In some embodiments, the colorant is a black colorant, such as a blackor dark colored ink, such as a black ink, dye component, orpigment-based composition. In some embodiments, the colorant is Indiaink, acid black 2, acid orange 7, direct Blue 14, or combinationthereof. Any suitable dye components can be selected depending on thefluorescent material used for the signal detection on the digitalmeasurement technique. For example, a combination of AO7 and DBu14reagents can be used as this combination may influence the fluoresceinsignal effectively in the femto-liter chambers. In some embodiments, adye combination can be selected to observe the target (favorable)emission signal for any assay (see e.g., FIG. 12).

i) India Ink

India ink (also known as Chinese ink) is a simple black or colored inkcomposed of a variety of fine soot, known as lampblack, combined withwater to form a liquid. The carbon molecules are in a colloidalsuspension and form a waterproof layer after drying. A binding agentsuch as gelatin or, more commonly, shellac may be added to make the inkmore durable once dried, however no binding agent is necessary. Indiaink may be in bottled form or solid form as an inkstick (most commonly,a stick), which must be ground and mixed with water before use. Indiaink may be waterproof or non-waterproof if a binder is used.

ii) Acid Black 2

Acid black 2, also known as nigrosin, is a dark black pigment (or dye)obtained primarily from aniline. The chemical structure of acid black 2is shown in FIG. 9B.

iii) Acid Orange 7

Acid orange 7 (“AO7”) also known as 2-naphthol orange, Orange II oracidic orange II, is a dye produced by a coupling reaction between2-Naphthol, or β-naphthol, and the diazonium compound of sulfanilicacid. The chemical structure of acid orange 7 is shown in FIG. 9C.

iv) Direct Blue 14

Direct Blue 14 (“DBU14”), also known as TRYPAN BLUE and VisionBlue, is adiazo-naphthalene sulfonate that is widely used as a stain. The chemicalstructure of Direct Blue 14 is shown in FIG. 9C.

b) “Addition” Method

The colorant can be used to reduce background fluorescence using an“Addition” method. The “Addition” method includes adding a colorant tothe aqueous phase or aqueous phase solution after the aqueous phase isdisplaced by sealant (e.g., oil) (i.e., the upper liquid phase includinga fluorescent substance), or adding a colorant to the sealant (e.g.,oil), to effectively reduce the background fluorescence noise in thefemto-liter chamber. The colorant suppresses the glow of the fluorescentsubstance in the upper liquid phase or aqueous phase. For example, a“Black Ink Addition” method involves adding black ink to the aqueousphase or aqueous phase solution after the displacement of the aqueousphase by the sealant (e.g., oil), or to the sealant (e.g., oil), thusthe time consuming step of removing the aqueous phase solution is notnecessary.

c) “Pre-Mix” Method

The colorant can be used to reduce background fluorescence using a“Pre-Mix” method (see FIG. 7A). The “Pre-Mix” method does not need anyadditional procedure after sealant (e.g., oil) sealing because theaqueous phase or aqueous phase solution in itself is a dark color, suchas a black color, thus the procedure is simplified and more efficient asonly inverting the sealant (e.g., oil) is needed. The colorant, such asblack ink, is added with a signal generating substrate to the aqueousphase or aqueous phase solution that include washed solid supportcomplexes, that includes a signal generating compound attached thereto,or added to an aqueous phase or aqueous phase solution containing thesignal generating compound or washed solid support complexes before thesignal generating substrate is added to the washed solid supportcomplexes. Pre-mixing the colorant, such as black ink, with the aqueousphase or aqueous phase solution could suppress the glow of fluoresceineven though the upper liquid phase (aqueous phase) includes thefluorescent substance. For example, a “Black Ink Pre-Mix” methodinvolves mixing black ink with the reacted signal generatingcompound/signal generating substrate (i.e., enzyme/substrate) solution,thus the time consuming step of removing the aqueous phase solution isnot necessary.

d) Sealant

In the present invention, the reaction vessels can be sealed or coveredby the addition of one or more solvents (“solvent well sealing”) using asealant, such as a hydrophilic or a hydrophobic solvent that has adensity that is heavier than the aqueous phase. Hydrophilic solventsthat can be used include hydrophilic alcohols, hydrophilic ethers,ketones, nitrile solvents, dimethyl sulfoxides, andN,N-dimethylformamides, or mixtures thereof. Examples of hydrophilicalcohols include ethanol, methanol, propanol, and glycerin. Examples ofhydrophilic ethers include tetrahydrofuran, polyethylene oxide, and1,4-dioxane. Examples of ketone include acetone and methyl ethyl ketone.Examples of the nitrile solvents include acetonitrile. Hydrophobicsolvents that can be used include hydrocarbons, unsaturatedhydrocarbons, aromatic hydrocarbons, silicone oils, perfluorocarbons,halogen solvents, hydrophobic ionic liquids and mixtures thereof.Examples of saturated hydrocarbons include alkanes, such as decane andhexadecane. Examples of unsaturated hydrocarbon include squalene.Examples of aromatic hydrocarbon include benzene and toluene. Examplesof perfluorocarbon encompass Fluorinert®, FC-40, FC-72, FC-84, FC-77,FC-3255, FC-3283, FC-43, FC-70), 3M Novec 4200, 3M Novec 4300, 3MFC-4432, 3M FC-4430, or 3M FC-4434. Examples of halogen solventsencompass chloroform, methylene chloride, and chlorobenzene. Thehydrophobic ionic liquid denotes ionic liquid which is not dissociatedat least in water. Examples of ionic liquids include1-butyl-3-methylimidazolium hexafluorophosphate.

e) Signal Generating Compound and Signal Generating Substrate

The detection of the analyte is correlated by the detectable product ordetectable label, namely, a signal, generated by the at least one signalgenerating compound and the at least one signal generating substrate. Insome embodiments, the at least one signal generating compound is anenzyme and the at least one signal generating substrate is a substratefor the enzyme. In some embodiments, the substrate for the enzyme is acolorimetric, fluorogenic (non-fluorescent) substrate or a chromogenicsubstrate. In some embodiments, the detectable signal is a fluorescentsignal. For example, the enzyme may be a, polynucleotidase, arginase,adenase, aminopolypeptidase, pepsin, lipases, catalase, tyrosinases,alcohol dehydrogenase, succinic dehydrogenase, diaphorase, glyoxalase,aldolase, glucose oxidase, horseradish peroxidase, galactosidase (suchas beta-galactosidase), phosphatases, phosphorylases and hexokinases orcombinations thereof. Examples of enzymatic substrates that can be usedinclude a chemiluminescent substrate such as CDP-Star®, (disodium4-chloro-3-(methoxyspiro{1,2-dioxetane-3,2′-(5′-chloro)tricyclo[3.3.1.1.s-up.3,7]decane}-4-yl)phenylphosphate), CSPD®, or (disodium3-(4-methoxyspiro{1,2-dioxetane-3,2-(5′-chloro)tricyclo[3.3.1.1-.sup.3,7]decane}-4-yl)phenylphosphate); a luminescent substrate such as p-nitrophenyl phosphate,5-bromo-4-chloro-3-indolyl phosphate (BCIP), 4-nitro blue tetrazoliumchloride (NBT), or iodonitrotetrazolium (INT); a fluorescent substratesuch as 4-methylumbelliferyl phosphate (4-MUP); and a chromogenicsubstrate such as 5-bromo-4-chloro-3-indolyl phosphate (BCIP), disodium5-bromo-6-chloro-indolyl phosphate, or p-nitrophenyl phosphate.

In some aspects, enzymes that can be used include those which contain aninhibitor molecule (such as a protein, peptide, etc.) bound to a siteother than the active binding site of the enzyme. Such inhibitormolecules change the conformation of the active binding site of theenzyme and prevent it from binding to the substrate. Examples ofinhibitor molecules include protease inhibitors. The inhibitor can beremoved from the enzyme using routine techniques known in the art toallow the enzyme to bind to the substrate thus allowing a signalgenerating reaction to occur.

In some embodiments, the enzyme can convert a non-fluorescent substrateinto a fluorescent substrate. In some embodiments, the enzyme cangenerate color using a chromogenic substrate.

3. Methods for Reducing Fluorescence Background Noise in a FluorescentDigital Immunoassay

The disclosed methods also relate to methods for reducing fluorescencebackground noise in a fluorescent digital immunoassay used to detect ananalyte in a sample, as described above. Solid phase material, such asCYTOP, cyclic olefin polymers (COP), and other resins, have some autofluorescence which affects the detection by increasing background noise.The disclosed methods use a digital counting device that includes ablack device. The black device includes a solid phase materialcomprising carbon black or a black film sheet attached to a transparentdevice. The solid phase material comprising carbon black or a black filmsheet can reduce the auto fluorescence. In some embodiments, the solidphase material is CYTOP, cyclic olefin polymers (COP), orpolydimethylsiloxane (PDMS). In some embodiments, the solid phasematerial includes CYTOP®, cyclic olefin polymers (COP),polydimethylsiloxane (PDMS), poly(methyl methacrylate), Polycarbonate(PC), or Polypropylene (PP). In some embodiments, the black device isthick enough so that excitation light to the nanowells and signal lightfrom the nanowells can be transmitted. In some embodiments, the devicethickness is between about 0.1 mm and about 1 mm, between about 0.5 mmand about 1 mm, about 0.1 mm and about 0.75 mm, about 0.5 mm and about0.75 mm. In some embodiments, the device thickness is less than about 1mm, less than about 0.9 mm, less than about 0.8 mm, less than about 0.7mm, less than about 0.6 mm, less than about 0.5 mm, less than about 0.4mm, less than about 0.3 mm, less than about 0.2 mm, or less than about0.1 mm.

In some embodiments, the method includes contacting a fluid samplecontaining or suspected of containing the analyte with a plurality ofsolid supports to create a mixture, wherein each solid support comprisesone or more first specific binding member capable of binding to theanalyte thereby producing solid support-first specific bindingmember-analyte complexes; washing the solid support-first specificbinding member-analyte complexes in the mixture with a first wash bufferto remove any solid support-first specific binding member not bound tothe analyte thereby producing a washed mixture; adding to the washedmixture one or more second specific binding members capable of bindingto the analyte thereby producing solid support-first specific bindingmember-analyte-second specific binding member complexes, wherein thesecond specific binding member comprises a signal generating compoundattached thereto; washing the solid support-first specific bindingmember-analyte-second specific binding member complexes with a secondwash buffer to remove any second specific binding member not bound tothe analyte bound to the first specific binding member thereby producingwashed solid support complexes; adding to the washed solid supportcomplexes a signal generating substrate, wherein the signal generatingcompound and the signal generating substrate produce a detectablesignal; spatially segregating at least a portion of the washed solidsupport complexes into a plurality of separate locations, wherein theplurality of separation locations comprises a plurality of reactionvessels and wherein the washed solid support complexes are present in anaqueous phase; covering the plurality of reaction vessels with a sealant(e.g., oil), wherein the aqueous phase is displaced by the sealant(e.g., oil) in the reaction chamber; and detecting the presence orabsence of the detectable signal using a digital counting device,wherein detection of the presence of the detectable signal indicates thepresence of a single molecule of analyte in the sample. In someembodiments, the method further includes adding a colorant, as describedabove. In some embodiments, the colorant is added to the washed solidsupport complexes simultaneously with the signal generating substrate,added to the washed solid support complexes before the signal generatingsubstrate, added to the washed solid support complexes after the signalgenerating substrate, added to the displaced aqueous phase, or added tothe sealant (e.g., oil), as described above.

4. Specific Binding Members

As will be appreciated by those in the art, the binding members will bedetermined by the analyte to be analyzed. Binding members for a widevariety of target molecules are known or can be readily found ordeveloped using known techniques. For example, when the target analyteis a protein, the binding members may include proteins, particularlyantibodies or fragments thereof (e.g., antigen-binding fragments (Fabs),Fab′ fragments, F(ab′)₂ fragments, recombinant antibodies, chimericantibodies, single-chain Fvs (“scFv”), single chain antibodies, singledomain antibodies, such as variable heavy chain domains (“VHH”; alsoknown as “VHH fragments”) derived from animals in the Camelidae family(VHH and methods of making them are described in Gottlin et al., Journalof Biomolecular Screening, 14:77-85 (2009)), recombinant VHHsingle-domain antibodies, and V_(NAR) fragments, disulfide-linked Fvs(“sdFv”), and anti-idiotypic (“anti-Id”) antibodies, and functionallyactive epitope-binding fragments of any of the above, full-lengthpolyclonal or monoclonal antibodies, antibody-like fragments, etc.),other proteins, such as receptor proteins, Protein A, Protein C, or thelike. In case where the analyte is a small molecule, such as, steroids,bilins, retinoids, and lipids, the first and/or the second specificbinding member may be a scaffold protein (e.g., lipocalins) or areceptor. In some cases, binding member for protein analytes may be apeptide. For example, when the target analyte is an enzyme, suitablebinding members may include enzyme substrates and/or enzyme inhibitorswhich may be a peptide, a small molecule and the like. In some cases,when the target analyte is a phosphorylated species, the binding membersmay comprise a phosphate-binding agent. For example, thephosphate-binding agent may comprise metal-ion affinity media such asthose describe in U.S. Pat. No. 7,070,921 and U.S. Patent ApplicationNo. 2006/0121544.

When the target molecule is a carbohydrate, potentially suitable capturecomponents (as defined herein) include, for example, antibodies,lectins, and selectins. As will be appreciated by those of ordinaryskill in the art, any molecule that can specifically associate with atarget molecule of interest may potentially be used as a binding member.

For certain embodiments, suitable target analyte/binding membercomplexes can include, but are not limited to, antibodies/antigens,antigens/antibodies, receptors/ligands, ligands/receptors,proteins/nucleic acid, enzymes/substrates and/or inhibitors,carbohydrates (including glycoproteins and glycolipids)/lectins and/orselectins, proteins/proteins, proteins/small molecules, etc.

In a particular embodiment, the first specific binding member and/orsecond specific binding member may be attached to a solid support via alinkage, which may comprise any moiety, functionalization, ormodification of the support and/or binding member that facilitates theattachment of the binding member to the support. The linkage between thebinding member and the support may include one or more chemical orphysical (e.g., non-specific attachment via van der Waals forces,hydrogen bonding, electrostatic interactions, hydrophobic/hydrophilicinteractions; etc.) bonds and/or chemical spacers providing suchbond(s).

In certain embodiments, a solid support may also comprise a protective,blocking, or passivating layer that can eliminate or minimizenon-specific attachment of non-capture components (e.g., analytemolecules, binding members) to the binding surface during the assaywhich may lead to false positive signals during detection or to loss ofsignal. Examples of materials that may be utilized in certainembodiments to form passivating layers include, but are not limited to:polymers, such as poly(ethylene glycol), that repel the non-specificbinding of proteins; naturally occurring proteins with this property,such as serum albumin and casein; surfactants, e.g., zwitterionicsurfactants, such as sulfobetaines; naturally occurring long-chainlipids; polymer brushes, and nucleic acids, such as salmon sperm DNA.

Certain embodiments utilize binding members that are proteins orpolypeptides. As is known in the art, any number of techniques may beused to attach a polypeptide to a wide variety of solid supports. A widevariety of techniques are known to add reactive moieties to proteins,for example, the method outlined in U.S. Pat. No. 5,620,850. Further,methods for attachment of proteins to surfaces are known, for example,see Heller, Acc. Chem. Res. 23:128 (1990).

As explained herein, binding between the binding members and theanalyte, is specific, e.g., as when the binding member and the analyteare complementary parts of a binding pair. In certain embodiments, thebinding member binds specifically to the analyte. By “specifically bind”or “binding specificity,” it is meant that the binding member binds theanalyte molecule with specificity sufficient to differentiate betweenthe analyte molecule and other components or contaminants of the testsample. For example, the binding member, according to one embodiment,may be an antibody that binds specifically to an epitope on an analyte.The antibody, according to one embodiment, can be any antibody capableof binding specifically to an analyte of interest. For example,appropriate antibodies include, but are not limited to, monoclonalantibodies, bispecific antibodies, minibodies, domain antibodies (dAbs)(e.g., such as described in Holt et al., (2014) Trends in Biotechnology21:484-490), and including single domain antibodies sdAbs that arenaturally occurring, e.g., as in cartilaginous fishes and camelid, orwhich are synthetic, e.g., nanobodies, VHH, or other domain structure),synthetic antibodies (sometimes referred to as antibody mimetics),chimeric antibodies, humanized antibodies, antibody fusions (sometimesreferred to as “antibody conjugates”), and fragments of each,respectively. As another example, the analyte molecule may be anantibody and the first specific binding member may be an antigen and thesecond specific binding member may be a secondary antibody thatspecifically binds to the target antibody or the first specific bindingmember may be a secondary antibody that specifically binds to the targetantibody and the second specific binding member may be an antigen.

In some embodiments, the binding member may be chemically programmedantibodies (cpAbs) (described in Rader (2014) Trends in Biotechnology32:186-197), bispecific cpAbs, antibody-recruiting molecules (ARMs)(described in McEnaney et al., (2012) ACS Chem. Biol. 7:1139-1151),branched capture agents, such as a triligand capture agent (described inMillward et al., (2011) J. Am. Chem. Soc. 133:18280-18288), engineeredbinding proteins derived from non-antibody scaffolds, such as monobodies(derived from the tenth fibronectin type III domain of humanfibronectin), affibodies (derived from the immunoglobulin bindingprotein A), DARPins (based on Ankyrin repeat modules), anticalins(derived from the lipocalins bilin-binding protein and human lipocalin2), and cysteine knot peptides (knottins) (described in Gilbreth andKoide, (2012) Current Opinion in Structural Biology 22:1-8; Banta etal., (2013) Annu. Rev. Biomed. Eng. 15:93-113),WW domains (described inPatel et al., (2013) Protein Engineering, Design & Selection26(4):307-314), repurposed receptor ligands, affitins (described inBéhar et al., (2013) 26:267-275), and/or Adhirons (described in Tiede etal., (2014) Protein Engineering, Design & Selection 27:145-155).

According to one embodiment in which an analyte is a biological cell(e.g., mammalian, avian, reptilian, other vertebrate, insect, yeast,bacterial, cell, etc.), the binding members may be ligands havingspecific affinity for a cell surface antigen (e.g., a cell surfacereceptor). In one embodiment, the binding member may be an adhesionmolecule receptor or portion thereof, which has binding specificity fora cell adhesion molecule expressed on the surface of a target cell type.In use, the adhesion molecule receptor binds with an adhesion moleculeon the extracellular surface of the target cell, thereby immobilizing orcapturing the cell, the bound cell may then be detected by using asecond specific binding member that may be the same as the firstspecific binding member or may bind to a different molecule expressed onthe surface of the cell.

In some embodiments, the binding affinity between analyte molecules andbinding members should be sufficient to remain bound under theconditions of the assay, including wash steps to remove molecules orparticles that are non-specifically bound. In some cases, for example inthe detection of certain biomolecules, the binding constant of theanalyte molecule to its complementary binding member may be between atleast about 10⁴ and about 10⁶M⁻¹, at least about 10⁵ and about 10⁹M⁻¹,at least about 10⁷ and about 10⁹M⁻¹, greater than about 10⁹ M⁻¹, orgreater.

5. Exemplary Target Analytes

As will be appreciated by those in the art, any analyte that can bespecifically bound by a first specific binding member and a secondspecific binding member may be detected and, optionally, quantifiedusing methods and devices of the present disclosure.

In some embodiments, the analyte may be a biomolecule or biologicalmolecule. Non-limiting examples of biomolecules and biological moleculesinclude macromolecules such as, proteins, lipids, and carbohydrates. Incertain instances, the analyte may be hormones, antibodies, growthfactors, cytokines, enzymes, receptors (e.g., neural, hormonal,nutrient, and cell surface receptors) or their ligands, cancer markers(e.g., PSA, TNF-alpha), markers of myocardial infarction (e.g.,troponin, creatine kinase, BNP, pro-BNP, NT-ProBNP, CK-MB, Galectin-3,and the like), thyroid markers (e.g., Anti-Tg, Anti-TPO, Free T3, FreeT4, T-uptake, Total T3, Total T4, TSH), toxins, drugs (e.g., drugs ofaddiction), metabolic agents (e.g., including vitamins), and the like.Non-limiting embodiments of protein analytes include peptides,polypeptides, protein fragments, protein complexes, fusion proteins,recombinant proteins, phosphoproteins, glycoproteins, lipoproteins, orthe like. In some embodiments, the analyte may be a biomarker, such as abiomarker for traumatic brain injury, sepsis, or coagulation, an analyteinvolved with general chemistry (e.g., ammonia, AST, cholesterol, etc.),a protein (e.g., transferrin, CRP, etc.), an analyte for therapeuticdrug monitoring (e.g., Methotrexate), an analyte for transplant (e.g.,tacrolimus), a drug of abuse, or a biomarker for genetic disorders.

In certain embodiments, the analyte may be a post-translationallymodified protein (e.g., phosphorylated, methylated, glycosylatedprotein) and the first or the second specific binding member may be anantibody specific to a post-translational modification. A modifiedprotein may be bound to a first specific binding member immobilized on asolid support where the first specific binding member binds to themodified protein but not the unmodified protein. In other embodiments,the first specific binding member may bind to both the unmodified andthe modified protein, and the second specific binding member may bespecific to the post-translationally modified protein.

In some embodiments, the analyte may be a cell, such as, circulatingtumor cell, pathogenic bacteria, viruses (including retroviruses,herpesviruses, adenoviruses, lentiviruses, Filoviruses (e.g., West Nile,Ebola and Zika viruses), hepatitis viruses (e.g., A, B, C, D, and E);HPV, Parvovirus, etc.; spores, etc.

A non-limiting list of analytes that may be analyzed by the methodspresented herein include Aβ42 amyloid beta-protein, fetuin-A, tau,secretogranin II, prion protein, Alpha-synuclein, tau protein,neurofilament light chain, parkin, PTEN induced putative kinase 1, DJ-1,leucine-rich repeat kinase 2, mutated ATP13A2, Apo H, ceruloplasmin,Peroxisome proliferator-activated receptor gamma coactivator-1 alpha(PGC-1α), transthyretin, Vitamin D-binding Protein, Active-B12, B12,cortisol, folate, frustosamine, homocysteine, intact PTH, pepsinogen I &II, DHEA-S, Estradiol, hCG, progesterone, prolactin, SHBG, testosterone,proapoptotic kinase R (PKR) and its phosphorylated PKR (pPKR), IL-12p40,CXCL13, IL-8, Dkk-3 (semen), p14 endocan fragment, Serum, ACE2,autoantibody to CD25, hTERT, CAI25 (MUC 16), VEGF, sIL-2, Osteopontin,Human epididymis protein 4 (HE4), Alpha-Fetoprotein, Albumin,albuminuria, microalbuminuria, neutrophil gelatinase-associatedlipocalin (NGAL), Cystatin C, interleukin 18 (IL-18), Kidney InjuryMolecule-1 (KIM-1), Liver Fatty Acid Binding Protein (L-FABP), LMP1,BARF1, IL-8, BRAF, CCNI, EGRF, FGF19, FRS2, GREB1, and LZTS1,alpha-amylase, carcinoembryonic antigen (CEA), CA 125, thioredoxin,beta-2 microglobulin levels—monitor activity of the virus, tumornecrosis factor-alpha receptors—monitor activity of the virus,Alpha-fetoprotein (AFP), CA15-3, CA 19-9, CYFRA 21-1, HE-4, PIVKA-11,ProGRP, SCC, follicle-stimulating hormone (FSH), leutinizing hormone(LH), T-cell lymphoma invasion and metastasis 1 (TIAM1), N-cadherin,EC39, amphiregulin, dUTPase, secretory gelsolin (pGSN), PSA (prostatespecific antigen), thymosin β15, insulin, plasma C-peptide, glycosylatedhemoglobin (HBA1c), C-Reactive Protein (CRP), Interleukin-6 (IL-6),ARHGDIB (Rho GDP-dissociation inhibitor 2), CFL1 (Cofilin-1), PFN1(profilin-1), GSTP1 (Glutathione S-transferase P), S100A11 (ProteinS100-A11), PRDX6 (Peroxiredoxin-6), HSPE1 (10 kDa heat shock protein,mitochondrial), LYZ (Lysozyme C precursor), GPI (Glucose-6-phosphateisomerase), HIST2H2AA (Histone H2A type 2-A), GAPDH(Glyceraldehyde-3-phosphate dehydrogenase), HSPG2 (Basementmembrane-specific heparan sulfate proteoglycan core protein precursor),LGALS3BP (Galectin-3-binding protein precursor), CTSD (Cathepsin Dprecursor), APOE (Apolipoprotein E precursor), IQGAP1 (RasGTPase-activating-like protein IQGAP1), CP (Ceruloplasmin precursor),and IGLC2 (IGLC1 protein), PCDGF/GP88, EGFR, HER2, MUC4, IGF-IR,p27(kip1), Akt, HER3, HER4, PTEN, PIK3CA, SHIP, Grb2, Gab2, PDK-1(3-phosphoinositide dependent protein kinase-1), TSC1, TSC2, mTOR, MIG-6(ERBB receptor feedback inhibitor 1), S6K, src, KRAS, MEKmitogen-activated protein kinase 1, cMYC, TOPO II topoisomerase (DNA) IIalpha 170 kDa, FRAP1, NRG1, ESR1, ESR2, PGR, CDKN1B, MAP2K1, NEDD4-1,FOXO3A, PPP1R1B, PXN, ELA2, CTNNB1, AR, EPHB2, KLF6, ANXA7, NKX3-1,PITX2, MKI67, PHLPP, adiponectin (ADIPOQ), fibrinogen alpha chain (FGA),leptin (LEP), advanced glycosylation end product-specific receptor (AGERaka RAGE), alpha-2-HS-glycoprotein (AHSG), angiogenin (ANG), CD14molecule (CD14), ferritin (FTH1), insulin-like growth factor bindingprotein 1 (IGFBP1), interleukin 2 receptor, alpha (IL2RA), vascular celladhesion molecule 1 (VCAM1) and Von Willebrand factor (VWF),myeloperoxidase (MPO), IL1α, TNFα, perinuclear anti-neutrophilcytoplasmic antibody (p-ANCA), lactoferrin, calprotectin, Wilm's Tumor-1protein, Aquaporin-1, MLL3, AMBP, VDAC1, E. coli enterotoxins(heat-labile exotoxin, heat-stable enterotoxin), influenza HA antigen,tetanus toxin, diphtheria toxin, botulinum toxins, Shiga toxin,Shiga-like toxin I, Shiga-like toxin II, Clostridium difficile toxins Aand B, etc.

Exemplary targets of may be measured in a sample such as anenvironmental sample, a biological sample obtained from a patient orsubject in need using the subject methods include: drugs of abuse (e.g.cocaine), protein biomarkers (including, but not limited to, Nucleolin,nuclear factor-kB essential modulator (NEMO), CD-30, protein tyrosinekinase 7 (PTK7), vascular endothelial growth factor (VEGF), MUC1glycoform, immunoglobulin μ Heavy Chains (IGHM), Immunoglobulin E, αvβ3integrin, α-thrombin, HIV gp120, NF-κB, E2F transcription factor, HER3,Plasminogen activator inhibitor, Tenascin C, CXCL12/SDF-1, prostatespecific membrane antigen (PSMA), gastric cancer cells, HGC-27; cells(including, but not limited to, non-small cell lung cancer (NSCLC),colorectal cancer cells, (DLD-1), H23 lung adenocarcinoma cells, Ramoscells, T-cell acute lymphoblastic leukemia (T-ALL) cells, CCRF-CEM,acute myeloid leukemia (AML) cells (HL60), small-cell lung cancer (SCLC)cells, NCIH69, human glioblastoma cells, U118-MG, PC-3 cells,HER-2-overexpressing human breast cancer cells, SK-BR-3, pancreaticcancer cell line (Mia-PaCa-2), and infectious agents (including, but notlimited to, Mycobacterium tuberculosis, Staphylococcus aureus, Shigelladysenteriae, Escherichia coli O157:H7, Campylobacter jejuni, Listeriamonocytogenes, Pseudomonas aeruginosa, Salmonella O8, and Salmonellaenteritidis).

Exemplary targets that may be measured in a sample obtained from apatient or subject in need using the subject methods include, but arenot limited to: HBV core capsid protein, CDK2, E2F transcription factor,Thymidylate synthase, Ras, EB1, and Receptor for Advanced Glycated Endproducts (RAGE).

6. Samples

As used herein, “sample”, “test sample”, “biological sample” refer tofluid sample containing or suspected of containing an analyte ofinterest. The sample may be derived from any suitable source. In somecases, the sample may comprise a liquid, fluent particulate solid, orfluid suspension of solid particles. In some cases, the sample may beprocessed prior to the analysis described herein. For example, thesample may be separated or purified from its source prior to analysis;however, in certain embodiments, an unprocessed sample containing theanalyte may be assayed directly. The source of the analyte molecule maybe synthetic (e.g., produced in a laboratory), the environment (e.g.,air, soil, fluid samples, e.g., water supplies, etc.), an animal, e.g.,a mammal, a plant, or any combination thereof. In a particular example,the source of an analyte is a human bodily substance (e.g., bodilyfluid, blood, serum, plasma, urine, saliva, sweat, sputum, semen, mucus,lacrimal fluid, lymph fluid, amniotic fluid, interstitial fluid, lunglavage, cerebrospinal fluid, feces, tissue, organ, or the like). Tissuesmay include, but are not limited to skeletal muscle tissue, livertissue, lung tissue, kidney tissue, myocardial tissue, brain tissue,bone marrow, cervix tissue, skin, etc. The sample may be a liquid sampleor a liquid extract of a solid sample. In certain cases, the source ofthe sample may be an organ or tissue, such as a biopsy sample, which maybe solubilized by tissue disintegration/cell lysis.

A wide range of volumes of the fluid sample may be analyzed. In a fewexemplary embodiments, the sample volume may be about 0.5 nL, about 1nL, about 3 nL, about 0.01 μL, about 0.1 μL, about 1 μL, about 5 μL,about 10 μL, about 100 μL, about 1 mL, about 5 mL, about 10 mL, or thelike. In some cases, the volume of the fluid sample is between about0.01 μL and about 10 mL, between about 0.01 μL and about 1 mL, betweenabout 0.01 μL and about 100 μL, or between about 0.1 μL and about 10 μL.

In some cases, the fluid sample may be diluted prior to use in an assay.For example, in embodiments where the source of an analyte molecule is ahuman body fluid (e.g., blood, serum), the fluid may be diluted with anappropriate solvent (e.g., a buffer such as PBS buffer). A fluid samplemay be diluted about 1-fold, about 2-fold, about 3-fold, about 4-fold,about 5-fold, about 6-fold, about 10-fold, about 100-fold, or greater,prior to use.

In some cases, the sample may undergo pre-analytical processing.Pre-analytical processing may offer additional functionality such asnonspecific protein removal and/or effective yet cheaply implementablemixing functionality. General methods of pre-analytical processing mayinclude the use of electrokinetic trapping, AC electrokinetics, surfaceacoustic waves, isotachophoresis, dielectrophoresis, electrophoresis, orother pre-concentration techniques known in the art. In some cases, thefluid sample may be concentrated prior to use in an assay. For example,in embodiments where the source of an analyte molecule is a human bodyfluid (e.g., blood, serum), the fluid may be concentrated byprecipitation, evaporation, filtration, centrifugation, or a combinationthereof. A fluid sample may be concentrated about 1-fold, about 2-fold,about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 10-fold,about 100-fold, or greater, prior to use.

In certain embodiments, the analyte is not amplified (i.e., the copynumber of the analyte is not increased) prior to the measurement of theanalyte. For example, in cases where the analyte is DNA or RNA, theanalyte is not replicated to increase copy numbers of the analyte. Incertain cases, the analyte is a protein or a small molecule.

7. Variations on Methods

The disclosed methods of determining the presence or amount of analyteof interest present in a sample may be as described above. The methodsmay also be adapted in view of other methods for analyzing analytes.Examples of well-known variations include, but are not limited to,immunoassay, such as sandwich immunoassay (e.g., monoclonal-polyclonalsandwich immunoassays, including enzyme detection (enzyme immunoassay(EIA) or enzyme-linked immunosorbent assay (ELISA), competitiveinhibition immunoassay (e.g., forward and reverse), enzyme multipliedimmunoassay technique (EMIT), a competitive binding assay,bioluminescence resonance energy transfer (BRET), one-step antibodydetection assay, homogeneous assay, heterogeneous assay, capture on thefly assay, etc. In some instances, the descriptions below may overlapthe method described above; in others, the descriptions below mayprovide alternates.

a) Immunoassay

The analyte of interest, and/or peptides or fragments thereof, may beanalyzed using an immunoassay. The presence or amount of analyte ofinterest can be determined using the herein-described antibodies anddetecting specific binding to analyte of interest. Any immunoassay maybe utilized. The immunoassay may be an enzyme-linked immunoassay(ELISA), a competitive inhibition assay, such as forward or reversecompetitive inhibition assays, or a competitive binding assay, forexample. In some embodiments, one signal generating compound or signalgenerating substrate is attached to the capture antibody and thedetection antibody. Alternately, a microparticle employed for capture,also can function for detection.

A homogeneous format may be used. For example, after the test sample isobtained from a subject, a mixture is prepared. The mixture contains thetest sample being assessed for analyte, a first specific bindingpartner, and a second specific binding partner. The order in which thetest sample, the first specific binding partner, and the second specificbinding partner are added to form the mixture is not critical. The testsample is simultaneously contacted with the first specific bindingpartner and the second specific binding partner. In some embodiments,the first specific binding partner and any analyte of interest containedin the test sample may form a first specific binding partner-analyte ofinterest-antigen complex and the second specific binding partner mayform a first specific binding partner-analyte of interest-secondspecific binding partner complex. In some embodiments, the secondspecific binding partner and any analyte of interest contained in thetest sample may form a second specific binding partner-analyte ofinterest-antigen complex and the first specific binding partner may forma first specific binding partner-analyte of interest-second specificbinding partner complex. Moreover, the second specific binding partneris labeled with or contains a detectable label as described herein.

A heterogeneous format may be used. For example, after the test sampleis obtained from a subject, a first mixture is prepared. The mixturecontains the test sample being assessed for analyte of interest and afirst specific binding partner, wherein the first specific bindingpartner and any analyte of interest contained in the test sample form afirst specific binding partner-analyte of interest complex. Preferably,the first specific binding partner is an anti-analyte of interestantibody or a fragment thereof. The order in which the test sample andthe first specific binding partner are added to form the mixture is notcritical. Preferably, the first specific binding partner is immobilizedon a solid phase. The solid phase used in the immunoassay (for the firstspecific binding partner and, optionally, the second specific bindingpartner) can be any solid phase known in the art, such as, but notlimited to, a magnetic particle, a bead a nanobead, a microbead, ananoparticle, a microparticle, a membrane, a scaffolding molecule, afilm, a filter paper, a disc, or a chip (e.g., a microfluidic chip). Inthose embodiments where the solid phase is a bead, the bead may be amagnetic bead or a magnetic particle. Magnetic beads/particles may beferromagnetic, ferrimagnetic, paramagnetic, superparamagnetic orferrofluidic. Exemplary ferromagnetic materials include Fe, Co, Ni, Gd,Dy, CrO₂, MnAs, MnBi, EuO, and NiO/Fe. Examples of ferrimagneticmaterials include NiFe₂O₄, CoFe₂O₄, Fe₃O₄ (or FeO.Fe₂O₃). Beads can havea solid core portion that is magnetic and is surrounded by one or morenon-magnetic layers. Alternately, the magnetic portion can be a layeraround a non-magnetic core. The solid support on which the firstspecific binding member is immobilized may be stored in dry form or in aliquid. The magnetic beads may be subjected to a magnetic field prior toor after contacting with the sample with a magnetic bead on which thefirst specific binding member is immobilized.

After the mixture containing the first specific binding partner-analyteof interest complex is formed, any unbound analyte of interest isremoved from the complex using any technique known in the art. Forexample, the unbound analyte of interest can be removed by washing.Desirably, however, the first specific binding partner is present inexcess of any analyte of interest present in the test sample, such thatall analyte of interest that is present in the test sample is bound bythe first specific binding partner.

After any unbound analyte of interest is removed, a second specificbinding partner is added to the mixture to form a first specific bindingpartner-analyte of interest-second specific binding partner complex. Thesecond specific binding partner is preferably an anti-analyte ofinterest antibody that binds to an epitope on analyte of interest thatdiffers from the epitope on analyte of interest bound by the firstspecific binding partner. Moreover, also preferably, the second specificbinding partner is labeled with or contains a signal generating compoundor signal generating substrate, as described above.

The use of immobilized antibodies or fragments thereof may beincorporated into the immunoassay. The antibodies may be immobilizedonto a variety of supports, such as magnetic or chromatographic matrixparticles, latex particles or modified surface latex particles, polymeror polymer film, plastic or plastic film, planar substrate, amicrofluidic surface, pieces of a solid substrate material, and thelike.

b) Sandwich Immunoassay

The sandwich immunoassay measures the amount of antigen between twolayers of antibodies (i.e., a capture antibody (i.e., at least onecapture antibody) and a detection antibody (i.e. at least one detectionantibody). The capture antibody and the detection antibody bind todifferent epitopes on the antigen, e.g., analyte of interest. Desirably,binding of the capture antibody to an epitope does not interfere withbinding of the detection antibody to an epitope. Either monoclonal orpolyclonal antibodies may be used as the capture and detectionantibodies in the sandwich immunoassay.

Generally, at least two antibodies are employed to separate and quantifyanalyte of interest in a test sample. More specifically, the at leasttwo antibodies bind to certain epitopes of analyte of interest or ananalyte of interest fragment forming an immune complex which is referredto as a “sandwich”. One or more antibodies can be used to capture theanalyte of interest in the test sample (these antibodies are frequentlyreferred to as a “capture” antibody or “capture” antibodies), and one ormore antibodies with a signal generating compound or signal generatingsubstrate that also bind the analyte of interest (these antibodies arefrequently referred to as the “detection” antibody or “detection”antibodies) can be used to complete the sandwich. In a sandwich assay,the binding of an antibody to its epitope desirably is not diminished bythe binding of any other antibody in the assay to its respectiveepitope. In other words, antibodies are selected so that the one or morefirst antibodies brought into contact with a test sample suspected ofcontaining analyte of interest do not bind to all or part of an epitoperecognized by the second or subsequent antibodies, thereby interferingwith the ability of the one or more second detection antibodies to bindto the analyte of interest. The capture antibody described above is anexample of a capture molecule. The detection antibody described above isan example of a detection molecule.

In a preferred embodiment, a test sample suspected of containing analyteof interest can be contacted with at least one capture antibody (orantibodies) and at least one detection antibodies either simultaneouslyor sequentially. In the sandwich assay format, a test sample suspectedof containing analyte of interest (membrane-associated analyte ofinterest, soluble analyte of interest, fragments of membrane-associatedanalyte of interest, fragments of soluble analyte of interest, variantsof analyte of interest (membrane-associated or soluble analyte ofinterest) or any combinations thereof) is first brought into contactwith the at least one capture antibody that specifically binds to aparticular epitope under conditions which allow the formation of anantibody-analyte of interest complex. If more than one capture antibodyis used, a multiple capture antibody-analyte of interest complex isformed. In a sandwich assay, the antibodies, preferably, the at leastone capture antibody, are used in molar excess amounts of the maximumamount of analyte of interest or the analyte of interest fragmentexpected in the test sample.

Optionally, prior to contacting the test sample with the at least onefirst capture antibody, the at least one capture antibody can be boundto a solid support which facilitates the separation the antibody-analyteof interest complex from the test sample. Any solid support known in theart can be used, including but not limited to, solid supports made outof polymeric materials in the form of planar substrates or beads, andthe like. The antibody (or antibodies) can be bound to the solid supportby adsorption, by covalent bonding using a chemical coupling agent or byother means known in the art, provided that such binding does notinterfere with the ability of the antibody to bind analyte of interestor analyte of interest fragment. Moreover, if necessary, the solidsupport can be derivatized to allow reactivity with various functionalgroups on the antibody. Such derivatization requires the use of certaincoupling agents such as, but not limited to, maleic anhydride,N-hydroxysuccinimide, azido, alkynyl, and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

After the test sample suspected of containing analyte of interest isbrought into contact with the at least one capture antibody, the testsample is incubated in order to allow for the formation of a captureantibody (or capture antibodies)-analyte of interest complex. Theincubation can be carried out at a pH of from about 4.5 to about 10.0,at a temperature of from about 2° C. to about 45° C., and for a periodfrom at least about one (1) minute to about eighteen (18) hours, fromabout 2-6 minutes, or from about 3-4 minutes.

After formation of the capture antibody (antibodies)-analyte of interestcomplex, the complex is then contacted with at least one detectionantibody (under conditions which allow for the formation of a captureantibody (antibodies)-analyte of interest-detection antibody(antibodies) complex). If the capture antibody-analyte of interestcomplex is contacted with more than one detection antibody, then acapture antibody (antibodies)-analyte of interest-detection antibody(antibodies) detection complex is formed. As with the capture antibody,when the at least one detection (and subsequent) antibody is broughtinto contact with the capture antibody-analyte of interest complex, aperiod of incubation under conditions similar to those described aboveis required for the formation of the capture antibody(antibodies)-analyte of interest-detection antibody (antibodies)complex. Preferably, at least one detection antibody contains a signalgenerating compound or signal generating substrate. The signalgenerating compound or signal generating substrate can be bound to theat least one detection antibody prior to, simultaneously with or afterthe formation of the capture antibody (antibodies)-analyte ofinterest-detection antibody (antibodies) complex.

The order in which the test sample and the specific binding partner(s)are added to form the mixture for assay is not critical. If the firstspecific binding partner is attached to the signal generating compoundor signal generating substrate, then signal generating compound orsignal generating substrate-attached first specific bindingpartner-analyte of interest complexes form. Alternatively, if a secondspecific binding partner is used and the second specific binding partneris attached to the signal generating compound or signal generatingsubstrate, then signal generating compound or signal generatingsubstrate-attached complexes of first specific binding partner-analyteof interest-second specific binding partner form. Any unbound specificbinding partner, whether labeled or unlabeled, can be removed from themixture using any technique known in the art, such as washing.

Next, signal, indicative of the presence of analyte of interest or afragment thereof is generated. Based on the parameters of the signalgenerated, the amount of analyte of interest in the sample can bequantified. Optionally, a standard curve can be generated using serialdilutions or solutions of known concentrations of analyte of interest bymass spectroscopy, gravimetric methods, and other techniques known inthe art.

c) Forward Competitive Inhibition

In a forward competitive format, an aliquot of labeled analyte ofinterest of a known concentration is used to compete with analyte ofinterest in a test sample for binding to analyte of interest antibody.

In a forward competition assay, an immobilized specific binding partner(such as an antibody) can either be sequentially or simultaneouslycontacted with the test sample and a labeled analyte of interest,analyte of interest fragment or analyte of interest variant thereof. Theanalyte of interest peptide, analyte of interest fragment or analyte ofinterest variant can be attached with a signal generating compound orsignal generating substrate. In this assay, the antibody can beimmobilized on to a solid support. Alternatively, the antibody can becoupled to an antibody, such as an antispecies antibody, that has beenimmobilized on a solid support, such as a microparticle or planarsubstrate.

The labeled analyte of interest, the test sample and the antibody areincubated under conditions similar to those described above inconnection with the sandwich assay format. Two different species ofantibody-analyte of interest complexes may then be generated.Specifically, one of the antibody-analyte of interest complexesgenerated contains a signal generating compound or signal generatingsubstrate while the other antibody-analyte of interest complex does notcontain a signal generating compound or signal generating substrate. Theantibody-analyte of interest complex can be, but does not have to be,separated from the remainder of the test sample prior to quantificationof the detectable product or detectable label. Regardless of whether theantibody-analyte of interest complex is separated from the remainder ofthe test sample, the amount of detectable product or detectable label(e.g., detectable signal) in the antibody-analyte of interest complex isthen quantified. The concentration of analyte of interest (such asmembrane-associated analyte of interest, soluble analyte of interest,fragments of soluble analyte of interest, variants of analyte ofinterest (membrane-associated or soluble analyte of interest) or anycombinations thereof) in the test sample can then be determined, e.g.,as described above. If helpful, determination can be done by comparingthe quantity of detectable product or detectable label (e.g., detectablesignal) in the antibody-analyte of interest complex to a standard curve.The standard curve can be generated using serial dilutions of analyte ofinterest (such as membrane-associated analyte of interest, solubleanalyte of interest, fragments of soluble analyte of interest, variantsof analyte of interest (membrane-associated or soluble analyte ofinterest) or any combinations thereof) of known concentration, whereconcentration is determined by mass spectroscopy, gravimetrically and byother techniques known in the art.

Optionally, the antibody-analyte of interest complex can be separatedfrom the test sample by binding the antibody to a solid support, such asthe solid supports discussed above in connection with the sandwich assayformat, and then removing the remainder of the test sample from contactwith the solid support.

d) Reverse Competition Assay

In a reverse competition assay, an immobilized analyte of interest caneither be sequentially or simultaneously contacted with a test sampleand at least one labeled antibody. The analyte of interest can be boundto a solid support, such as the solid supports discussed above inconnection with the sandwich assay format.

The immobilized analyte of interest, test sample and at least onelabeled antibody are incubated under conditions similar to thosedescribed above in connection with the sandwich assay format. Twodifferent species analyte of interest-antibody complexes are thengenerated. Specifically, one of the analyte of interest-antibodycomplexes generated is immobilized and contains a signal generatingcompound or signal generating substrate while the other analyte ofinterest-antibody complex is not immobilized and contains signalgenerating compound or signal generating substrate. The non-immobilizedanalyte of interest-antibody complex and the remainder of the testsample are removed from the presence of the immobilized analyte ofinterest-antibody complex through techniques known in the art, such aswashing. Once the non-immobilized analyte of interest antibody complexis removed, the amount of signal generating compound or signalgenerating substrate in the immobilized analyte of interest-antibodycomplex is then quantified. The concentration of analyte of interest inthe test sample can then be determined by comparing the quantity ofdetectable signal as described above. If helpful, this can be done withuse of a standard curve. The standard curve can be generated usingserial dilutions of analyte of interest or analyte of interest fragmentof known concentration, where concentration is determined by massspectroscopy, gravimetrically and by other techniques known in the art.

e) One-Step Immunoassay or Capture on the Fly Assay

In a one-step immunoassay or capture on the fly assay, a solid substrateis pre-coated with an immobilization agent. The capture agent, theanalyte and the detection agent are added to the solid substratetogether, followed by a wash step prior to detection. The capture agentcan bind the analyte and comprises a ligand for an immobilization agent.The capture agent and the detection agents may be antibodies or anyother moiety capable of capture or detection as described herein orknown in the art. The ligand may comprise a peptide tag and animmobilization agent may comprise an anti-peptide tag antibody.Alternately, the ligand and the immobilization agent may be any pair ofagents capable of binding together so as to be employed for a capture onthe fly assay (e.g., specific binding pair, and others such as are knownin the art). More than one analyte may be measured. In some embodiments,the solid substrate may be coated with an antigen and the analyte to beanalyzed is an antibody.

In some embodiments, a solid support (such as a microparticle)pre-coated with an immobilization agent (such as biotin, streptavidin,etc.) and at least a first specific binding member and a second specificbinding member (which function as capture and detection reagents,respectively) are used. The first specific binding member comprises aligand for the immobilization agent (for example, if the immobilizationagent on the solid support is streptavidin, the ligand on the firstspecific binding member may be biotin) and also binds to the analyte ofinterest. The second specific binding member comprises a signalgenerating compound or signal generating substrate and binds to ananalyte of interest. The solid support and the first and second specificbinding members may be added to a test sample (either sequentially orsimultaneously). The ligand on the first specific binding member bindsto the immobilization agent on the solid support to form a solidsupport/first specific binding member complex. Any analyte of interestpresent in the sample binds to the solid support/first specific bindingmember complex to form a solid support/first specific bindingmember/analyte complex. The second specific binding member binds to thesolid support/first specific binding member/analyte complex and thesignal generating compounds or signal generating substrates detected. Anoptional wash step may be employed before the detection. In certainembodiments, in a one-step assay more than one analyte may be measured.In certain other embodiments, more than two specific binding members canbe employed. In certain other embodiments, multiple signal generatingcompounds or signal generating substrates can be added. In certain otherembodiments, multiple analytes of interest can be detected.

The use of a one step immunoassay or capture on the fly assay can bedone in a variety of formats as described herein, and known in the art.For example the format can be a sandwich assay such as described above,but alternately can be a competition assay, can employ a single specificbinding member, or use other variations such as are known.

f) Combination Assays (Co-Coating of Microparticles with Ag/Ab)

In a combination assay, a solid substrate, such as a microparticle isco-coated with an antigen and an antibody to capture an antibody and anantigen from a sample, respectively. The solid support may be co-coatedwith two or more different antigens to capture two or more differentantibodies from a sample. The solid support may be co-coated with two ormore different antibodies to capture two or more different antigens froma sample.

Additionally, the methods described herein may use blocking agents toprevent either specific or non-specific binding reactions (e.g., HAMAconcern) among assay compounds. Once the agent (and optionally, anycontrols) is immobilized on the support, the remaining binding sites ofthe agent may be blocked on the support. Any suitable blocking reagentknown to those of ordinary skill in the art may be used. For example,bovine serum albumin (“BSA”), phosphate buffered saline (“PBS”)solutions of casein in PBS, Tween 20™ (Sigma Chemical Company, St.Louis, Mo.), or other suitable surfactant, as well as other blockingreagents, may be employed.

As is apparent from the present disclosure, the methods disclosedherein, including variations, may be used for diagnosing a disease,disorder or condition in a subject suspected of having the disease,disorder, or condition. For example, the sample analysis may be usefulfor detecting a disease marker, such as, a cancer marker, a marker for acardiac condition, a toxin, a pathogen, such as, a virus, a bacteria, ora portion thereof. The methods also may be used for measuring analytepresent in a biological sample. The methods also may be used in bloodscreening assays to detect a target analyte. The blood screening assaysmay be used to screen a blood supply.

8. Multiplexing

The methods may include one or more (or alternately two or more)specific binding members to detect one or more (or alternately two ormore) target analytes in the sample in a multiplexing assay. Each of theone or more (or alternately two or more) specific binding members bindsto a different target analyte and each specific binding member isconjugated to a different signal generating compound or signal generatedsubstrate. For example, a first specific binding member binds to a firsttarget analyte, a second specific binding member binds to a secondtarget analyte, a third specific binding member binds to a third targetanalyte, etc. and the first specific binding member is labeled with afirst signal generating compound or first signal generating substrate,the second specific binding member is labeled with a second signalgenerating compound or second signal generating substrate, the thirdspecific binding member is labeled with a third signal generatingcompound or a third signal generating substrate, etc. In someembodiments, the conditions of the sample can be changed at varioustimes during the assay, allowing detection of the first signalgenerating compound or first signal generating substrate, the secondsignal generating compound or second signal generating substrate, thethird signal generating compound or third signal generating substrate,etc., thereby detecting one or more (or alternately two or more) targetanalytes. In some embodiments, the one or more (or alternately two ormore) signal generating compounds or signal generating substrates aredetected simultaneously. In some embodiments, the one or more (oralternately two or more) signal generating compounds or signalgenerating substrates are detected consecutively. In some embodiments,the one or more (or alternately two or more) signal generating compoundsor signal generating substrates generates a different detectable signal,such as a different wavelength of fluorescence signal.

Alternatively, each of the one or more (or alternately two or more)specific binding members binds to a different target analyte and eachspecific binding member is conjugated to a different solid support, suchas a different fluorophore bead. For example, a first specific bindingmember binds to a first target analyte, a second specific binding memberbinds to a second target analyte, a third specific binding member bindsto a third target analyte, etc., the first specific binding member islabeled with a first signal generating compound or first signalgenerating substrate, the second specific binding member is labeled witha second signal generating compound or second signal generatingsubstrate, the third specific binding member is labeled with a thirdsignal generating compound or a third signal generating substrate, etc.,and the first specific binding member is immobilized on a first solidsupport, the second specific binding member is immobilized on a secondsolid support, the third specific binding member is immobilized on athird solid support, etc. In some embodiments, the one or more (oralternately two or more) signal generating compounds or signalgenerating substrates generates a different detectable signal, such as adifferent wavelength or fluorescence signal, and the different solidsupports is detected simultaneously or consecutively.

In some embodiments, a first specific binding member binds to a firsttarget analyte, a second specific binding member binds to a secondtarget analyte, a third specific binding member binds to a third targetanalyte, etc., the first specific binding member, the second specificbinding member, the third specific binding member, etc. are labeled witha signal generating compound or a signal generating substrate, and thefirst specific binding member is immobilized on a first solid support,the second specific binding member is immobilized on a second solidsupport, the third specific binding member is immobilized on a thirdsolid support, etc. In some embodiments, the signal generating compoundsor signal generating substrates generates a detectable signal, such as adifferent wavelength or fluorescence signal, and the different solidsupports is detected simultaneously or consecutively.

9. Kits

Also provided herein is a kit for use in performing the above-describedmethods. The kit may include instructions for analyzing the analyte withthe disclosed methods. Instructions included in the kit may be affixedto packaging material or may be included as a package insert. Theinstructions may be written or printed materials, but are not limited tosuch. Any medium capable of storing such instructions and communicatingthem to an end user is contemplated by this disclosure. Such mediainclude, but are not limited to, electronic storage media (e.g.,magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM),and the like. As used herein, “instructions” may include the address ofan internet site that provides the instructions.

Alternatively or additionally, the kit may comprise a calibrator orcontrol, e.g., purified, and optionally lyophilized analyte of interestor in liquid, gel or other forms, and/or at least one container (e.g.,tube, microtiter plates or strips) for use with the methods describedabove, and/or a buffer, such as an assay buffer or a wash buffer, eitherone of which can be provided as a concentrated solution. In someembodiments, the kit comprises all components, i.e., reagents,standards, buffers, diluents, etc., which are necessary to perform theassay. The instructions also can include instructions for generating astandard curve.

The kit may further comprise reference standards for quantifying theanalyte of interest. The reference standards may be employed toestablish standard curves for interpolation and/or extrapolation of theanalyte of interest concentrations. The kit may include referencestandards that vary in terms of concentration level. For example, thekit may include one or more reference standards with either a highconcentration level, a medium concentration level, or a lowconcentration level. In terms of ranges of concentrations for thereference standard, this can be optimized per the assay. Exemplaryconcentration ranges for the reference standards include but are notlimited to, for example: about 10 fg/mL, about 20 fg/mL, about 50 fg/mL,about 75 fg/mL, about 100 fg/mL, about 150 fg/mL, about 200 fg/mL, about250 fg/mL, about 500 fg/mL, about 750 fg/mL, about 1000 fg/mL, about 10pg/mL, about 20 pg/mL, about 50 pg/mL, about 75 pg/mL, about 100 pg/mL,about 150 pg/mL, about 200 pg/mL, about 250 pg/mL, about 500 pg/mL,about 750 pg/mL, about 1 ng/mL, about 5 ng/mL, about 10 ng/mL, about12.5 ng/mL, about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 40ng/mL, about 45 ng/mL, about 50 ng/mL, about 55 ng/mL, about 60 ng/mL,about 75 ng/mL, about 80 ng/mL, about 85 ng/mL, about 90 ng/mL, about 95ng/mL, about 100 ng/mL, about 125 ng/mL, about 150 ng/mL, about 165ng/mL, about 175 ng/mL, about 200 ng/mL, about 225 ng/mL, about 250ng/mL, about 275 ng/mL, about 300 ng/mL, about 400 ng/mL, about 425ng/mL, about 450 ng/mL, about 465 ng/mL, about 475 ng/mL, about 500ng/mL, about 525 ng/mL, about 550 ng/mL, about 575 ng/mL, about 600ng/mL, about 700 ng/mL, about 725 ng/mL, about 750 ng/mL, about 765ng/mL, about 775 ng/mL, about 800 ng/mL, about 825 ng/mL, about 850ng/mL, about 875 ng/mL, about 900 ng/mL, about 925 ng/mL, about 950ng/mL, about 975 ng/mL, about 1000 ng/mL, about 2 μg/mL, about 3 μg/mL,about 4 μg/mL, about 5 μg/mL, about 6 μg/mL, about 7 μg/mL, about 8μg/mL, about 9 μg/mL, about 10 μg/mL, about 20 μg/mL, about 30 μg/mL,about 40 μg/mL, about 50 μg/mL, about 60 μg/mL, about 70 μg/mL, about 80μg/mL, about 90 μg/mL, about 100 μg/mL, about 200 μg/mL, about 300μg/mL, about 400 μg/mL, about 500 μg/mL, about 600 μg/mL, about 700μg/mL, about 800 μg/mL, about 900 μg/mL, about 1000 μg/mL, about 2000μg/mL, about 3000 μg/mL, about 4000 μg/mL, about 5000 μg/mL, about 6000μg/mL, about 7000 μg/mL, about 8000 μg/mL, about 9000 μg/mL, or about10000 μg/mL.

Any specific binding members, which are provided in the kit mayincorporate an at least one signal generating compound, one or moresignal generating substrates, or the like, or the kit can includereagents for labeling the specific binding members or reagents fordetecting the specific binding members and/or for labeling the analytesor reagents for detecting the analyte. If desired, the kit can containone or more different signal generating compounds and/or signalgenerating or substrates. The specific binding members, calibrators,and/or controls can be provided in separate containers or pre-dispensedinto an appropriate assay format.

The kit may include one or more specific binding members, for example,to detect one or more target analytes in the sample in a multiplexingassay. The number of different types of specific binding members in thekit may range widely depending on the intended use of the kit. Thenumber of specific binding members in the kit may range from 1 to about10, or higher. For example, the kit may include 1 to 10 specific bindingmembers, 1 to 9 specific binding members, 1 to 8 specific bindingmembers, 1 to 7 specific binding members, 1 to 6 specific bindingmembers, 1 to 5 specific binding members, 1 to 4 specific bindingmembers, 1 to 3 specific binding members, 1 to 2 specific bindingmembers, 2 to 10 specific binding members, 2 to 9 specific bindingmembers, 2 to 8 specific binding members, 2 to 7 specific bindingmembers, 2 to 6 specific binding members, 2 to 5 specific bindingmembers, 2 to 4 specific binding members, 3 to 10 specific bindingmembers, 3 to 9 specific binding members, 3 to 8 specific bindingmembers, 3 to 7 specific binding members, 3 to 6 specific bindingmembers, 3 to 5 specific binding members, 3 to 4 specific bindingmembers, 4 to 10 specific binding members, 4 to 9 specific bindingmembers, 4 to 8 specific binding members, 4 to 7 specific bindingmembers, 4 to 6 specific binding members, 5 to 10 specific bindingmembers, 5 to 9 specific binding members, 5 to 8 specific bindingmembers, 5 to 7 specific binding members, 5 to 6 specific bindingmembers, 6 to 10 specific binding members, 6 to 9 specific bindingmembers, 6 to 8 specific binding members, 6 to 7 specific bindingmembers, 7 to 10 specific binding members, 7 to 9 specific bindingmembers, 7 to 8 specific binding members, 8 to 10 specific bindingmembers, 8 to 9 specific binding members, or 9 to 10 specific bindingmembers. Each of the one or more specific binding members may bind to adifferent target analyte and each specific binding member may beassociated with a different signal generating compound and/or signalgenerating substrate. For example, the kit may include a first specificbinding member that binds to a first target analyte, a second specificbinding member that binds to a second target analyte, a third specificbinding member that binds to a third target analyte, etc. and the firstspecific binding member is associated with a first signal generatingcompound and/or first signal generating substrate, the second specificbinding member is associated with a second signal generating compoundand/or second signal generating substrate, the third specific bindingmember is associated with a third signal generating compound and/orthird signal generating substrate, etc. In addition to the one or morespecific binding members, the kits may further comprise one or moreadditional assay components, such as suitable buffer media, and thelike. The kits may also include a device for detecting and measuring thesignal generating compound and/or signal generating substrate, such asthose described supra. Finally, the kits may comprise instructions forusing the specific binding members in methods of analyte detectionaccording to the subject invention, where these instructions for use maybe present on the kit packaging and/or on a package insert.

Optionally, the kit includes quality control components (for example,sensitivity panels, calibrators, and positive controls). Preparation ofquality control reagents is well-known in the art and is described oninsert sheets for a variety of immunodiagnostic products. Sensitivitypanel members optionally are used to establish assay performancecharacteristics, and further optionally are useful indicators of theintegrity of the kit reagents, and the standardization of assays.

The kit can also optionally include other reagents required to conduct adiagnostic assay or facilitate quality control evaluations, such asbuffers, salts, enzymes, enzyme co-factors, substrates, detectionreagents, and the like. Other components, such as buffers and solutionsfor the isolation and/or treatment of a test sample (e.g., pretreatmentreagents), also can be included in the kit. The kit can additionallyinclude one or more other controls. One or more of the components of thekit can be lyophilized, in which case the kit can further comprisereagents suitable for the reconstitution of the lyophilized components.One or more of the components may be in liquid form.

The various components of the kit optionally are provided in suitablecontainers as necessary. The kit further can include containers forholding or storing a sample (e.g., a container for a urine, saliva,plasma, cerebrospinal fluid, or serum sample, or appropriate containerfor storing, transporting or processing tissue so as to create a tissueaspirate). Where appropriate, the kit optionally also can containreaction vessels, mixing vessels, and other components that facilitatethe preparation of reagents or the test sample. The kit can also includeone or more sample collection/acquisition instruments for assisting withobtaining a test sample, such as various blood collection/transferdevices such as microsampling devices, micro-needles, or other minimallyinvasive pain-free blood collection methods; blood collection tube(s);lancets; capillary blood collection tubes; other single fingertip-prickblood collection methods; buccal swabs, nasal/throat swabs; 16-gauge orother size needle, circular blade for punch biopsy (e.g., 1-8 mm, orother appropriate size), surgical knife or laser (e.g., particularlyhand-held), syringes, sterile container, or canula, for obtaining,storing or aspirating tissue samples; or the like. The kit can includeone or more instruments for assisting with joint aspiration, conebiopsies, punch biopsies, fine-needle aspiration biopsies, image-guidedpercutaneous needle aspiration biopsy, bronchoaveolar lavage, endoscopicbiopsies, and laproscopic biopsies.

If desired, the kit can contain a solid phase, such as a magneticparticle, bead, membrane, scaffolding molecule, film, filter paper,disc, or chip.

If desired, the kit can further comprise one or more components, aloneor in further combination with instructions, for assaying the testsample for another analyte, which can be a biomarker, such as abiomarker of a disease state or disorder, such as infectious disease,cardiac disease, metabolic disease, thyroid disease, etc.

The present invention has multiple aspects, illustrated by the followingnon-limiting examples.

10. EXAMPLES Example 1 “Black Ink Addition” Method

The “Black Ink Addition” method, shown in FIG. 3, involves the additionof black ink, such as India Ink, into the aqueous phase (the upperliquid phase including the fluorescent substrate) after the displacementof the aqueous phase by the sealant (e.g., oil) (see step 7 of FIG. 3).The substrate for the assay was fluorescein-di-phosphate (FDP). Thebuffer for the enzymatic reaction was 1M diethanolamine (DEA), 1 mMMgCl₂, 0.05% Tween20, and 300 μM FDP, pH 9.25. The digital ELISA usingthe “Black Ink Addition” method, in which black ink was added to theaqueous phase and the aqueous phase was not removed (C; see FIG. 3), wascompared with digital ELISA that did not remove the aqueous phase (A)and digital ELISA that removed the aqueous phase (B). The number oftarget molecules was determined by counting the number of fluorescentdroplets under an optical microscope using a fluorescein filter. At thesame time, the total number of beads was counted using atetramethylrhodamine (TRITC) filter. The digital ELISA with the “BlackInk Addition” method showed an improvement for counting the number offluorescent droplets and increased number of analyzable images (FIGS. 4and 5 and Table 1).

TABLE 1 Analyzable Image Aqueous Phase (# of pieces/45 pieces) Aremained with fluorescein 0 B removed sometime some remaining 29 withfluorescein C remained with fluorescein and black 44 ink

As shown in FIG. 4, both the TRITC and fluorescein filtered images wereclear even if black ink was added. Fluorescent substance present in theupper aqueous phase obstructed the counting of the number of fluorescentdroplets under an optical microscope. There was no fluorescein data for“A” (FIG. 4 and Table 3), in which the aqueous phase was not removed,due to the strong brightness of the fluorescent substance present in theupper aqueous phase. Only 29 pieces of analyzable images were obtainedfor “B” in which the upper aqueous solution was removed manually (FIG. 4and Table 3), due to the fluorescent substance present in incompleteremoval of the upper aqueous phase which resulted in some upper aqueousphase remaining in the well that obstructed the counting of the numberof fluorescent droplets in 16 pieces of data. In contrast, 44 out of 45images were acquired using the “Black Ink Addition” method without theremoval of the aqueous phase.

There were 2 patterns of the glowed image in the acquired image for the16 pieces of data that were unanalyzable for “B” (FIG. 6—top left andtop right images). One pattern (15 out of 16 pieces) showed a highbackground image with the fluorescein filter due to glowing widely (topleft images), and the other pattern (1 out of 16 pieces) showed an theimage that strongly glowed around an edge (top right images). In thebottom images of FIG. 6, black ink was added into the sealant (e.g.,oil) of the device well containing the fluorescent reaction. In thebottom left image, the high background due to the incomplete removal ofthe upper aqueous phase disappeared by adding the black ink as the 15pieces that had high background image with the fluorescein filter showedclearer images, indicating that adding black ink can reduce backgroundfluorescence even if the manual removal of the upper aqueous solution isincomplete. On the other hand, the right image pattern seen in the onepiece that had an image that strongly glowed around an edge could not beanalyzed even if black ink was added, as this glow signal came fromresidual aqueous phase on the device side (bottom side of the devicewell).

Example 2 “Black Ink Pre-Mix” Method

To solve the issue of the time consuming step of removing the aqueousphase, which can be incomplete, the “Black Ink Pre-Mix X” method wasdeveloped. As shown in FIG. 7A, the “Black Ink Pre-Mix” method involvesthe addition of black ink, such as India Ink, to the solution of theenzyme reaction before adding the enzyme reaction to the device well.The digital ELISA with the “Black Ink Pre-Mix” method (i.e., black inkwas added to the substrate solution) showed an improved counting of thenumber of fluorescent droplets and increased analyzable images, ascompared with the “Black Ink Addition” method. Table 2 shows theexperimental conditions and the final concentration of black ink in eachsample. The substrate for the assay was fluorescein-di-phosphate (FDP).The buffer for the enzymatic reaction was 1M diethanolamine (DEA), 1 mMMgCl₂, 0.05% Tween20, and 300 μM FDP, pH 9.25. As shown in FIG. 8, thesignal % levels were almost same indicating that all images could beanalyzed by the “Black Ink Pre-Mix” method with little or no affect onthe detection of the true signal (Signal %).

TABLE 2 Methods Final Conc. Of Ink A Remove Aqueous Phase  0% B ADDITION10% C PRE-MIX 3.3%  D 6.7%  E 10% F 13%

For the sample treated with the “Black Ink Addition” method(“ADDITION”), some images could not be analyzed because the glow signaloriginated from the residual aqueous phase on the device side (bottomside of the device well). In the samples treated with the “Black InkPre-Mix” method, the glow of the fluorescence in the aqueous phase wassuppressed no matter regardless of whether aqueous phase was present ornot. Therefore, all images were analyzed completely because the glowedsignal was eliminated by the black ink/substrate solution. Furthermore3.3%, 6.7%, 10% of black ink with the “Black Ink Pre-Mix IX” methodobtained similar results to the 10% result of the “Black Ink Addition”method (see FIG. 8). These result showed that black ink did not affectthe enzyme reaction in the solution. In addition, the true signals couldbe observed even if the background was completely black.

Example 3 Other Dyes

Acid Black 2 (“ABk2”), another dye component, was used to reducebackground glow noise in both “Black Ink Addition” and “Black InkPre-Mix” methods. 10% India Ink and 25 mM Acid Black 2 were compared andhad similar images for signal detection with threshold optimization. SeeFIG. 10 and Table 3. When the images of the digital assay were analyzed,a bright spot was used as a threshold for detection. All pixels thatmade up an image had unique intensity values corresponding with thebrightness. The dark pixels had lower values than bright pixels. Athreshold was set to determine if a pixel was bright or dark(binarization). When using the black ink method, an appropriatethreshold was different against previous assays, thus a new thresholdwas set (“threshold optimization”). Both India Ink (“Black Ink”) andAcid Black 2 (“ABk2”) effectively reduced the background fluorescence(“glow noise”) using the “Black Ink Addition” or “Black Ink Pre-mix”method.

TABLE 3 Method Sample (Threshold) Sample Average % of Signal A Addition10% black ink 0 fM 0.020 (700/1000) 10 fM 5.008 B 25 mM ABk2 0 fM 0.014(700/1000) 10 fM 5.689 C Pre-Mix 10% black ink 0 fM 0.031 (700/1000) 10fM 3.929 D 25 mM ABk2 0 fM 0.027 (1100/450) 10 fM 5.523 E 25 mM ABk2 0fM 0.016 (1100/500) 10 fM 4.222

Other dyes, Acid Orange 7 (AO7) and Direct Blue 14 (DBu14) (see FIGS. 9Aand 9B), were combined and compared with black ink, i.e., ABk2. SeeTable 4 and FIG. 11. The combination of dyes reduced background glownoise in the “Black Ink Addition” or “Black Ink Pre-mix” method.

TABLE 4 Average Method Dye Additive Sample % of Signal % CV A Addition10% black ink 0 fM 0.092 32.17 10 fM 6.520 2.64 B 15 mM AO7 0 fM 0.11312.69 4 mM DBu14 10 fM 6.492 4.77 C Pre-Mix 10% black ink 0 fM 0.06719.11 10 fM 5.953 2.25 D 5 mM AO7 0 fM 0.070 21.40 1 mM DBu14 10 fM 5.782.36

The combination of 15 mM Acid Orange 7 (AO7) and 4 mM Direct Blue 14(DBu14) reduced the background glow noise in the “Black Ink Addition.”See FIG. 11. The combination of 5 mM AO7 and 1 mM DBu14 reduced thebackground glow noise in the “Black Ink Pre-Mix” method. Higherconcentrations of AO7 and DBu14 (15 mM and 4 mM, respectively) with the“Black Ink Pre-Mix” method also reduced the background glow noise butthe positive signal with 10 fM antigen was low.

Example 4 Detection of Low Concentration of Antigen

The detection ability of the “Black Ink Addition” and “Black InkPre-Mix” methods were evaluated using a low concentration of antigen,0.1 fM sample (see Table 5 and FIG. 13).

TABLE 5 0 fM 0.1 fM % Signal SD % Signal SD S/N ratio 25 mM ABk2 0.0130.010 0.056 0.014 4.4 25 mM ABk2 0.015 0.002 0.047 0.009 3.1 5 mM AO7 +0.026 0.002 0.051 0.018 1.9 1 mM DBu14

The signal to noise (S/N) ratios for the 0.1 fM sample detection were4.4 for ABk2 using the “Black Ink Addition” method, 3.1 for ABk2 usingthe “Black Ink Pre-Mix MIX” method, and 1.9 for the combination of AO7and DBu14 sing the “Black Ink Pre-Mix” method.

Example 5 Black Device

A “Black Device” can be prepared to reduce background glow noise (seeFIG. 14) as an alternative to adding black ink to the digital assay. Theleft image of FIG. 14 shows the current device using a transparentprepared CYTOP on glass or COP, PDMS polymer. The right image of FIG. 14shows the addition of black dye or material to the device, e.g. carbonblack into CYTOP or polymer, or black film sheet attaching a transparentdevice for the background glow noise reduction.

Finally, although the various aspects and features of the invention havebeen described with respect to various embodiments and specific examplesherein, all of which may be made or carried out conventionally, it willbe understood that the invention is entitled to protection within thefull scope of the appended claims.

It is understood that the foregoing detailed description andaccompanying examples are merely illustrative and are not to be taken aslimitations upon the scope of the invention, which is defined solely bythe appended claims and their equivalents.

Various changes and modifications to the disclosed embodiments will beapparent to those skilled in the art. Such changes and modifications,including without limitation those relating to the chemical structures,substituents, derivatives, intermediates, syntheses, compositions,formulations, or methods of use of the invention, may be made withoutdeparting from the spirit and scope thereof.

For reasons of completeness, various aspects of the invention are setout in the following numbered clause:

Clause 1. A signal-generating digital assay for determining the presenceor absence of a single molecule of an analyte in a fluid sample, themethod comprising: (a) contacting the fluid sample containing orsuspected of containing the analyte with a plurality of solid supportsto create a mixture, wherein each solid support comprises one or morefirst specific binding member capable of binding to the analyte therebyproducing solid support-first specific binding member-analyte complexes;(b) washing the solid support-first specific binding member-analytecomplexes in the mixture with a first wash buffer to remove any solidsupport-first specific binding member not bound to the analyte therebyproducing a washed mixture; (c) adding to the washed mixture one or moresecond specific binding members capable of binding to the analytethereby producing solid support-first specific bindingmember-analyte-second specific binding member complexes, wherein thesecond specific binding member comprises a signal generating compoundattached thereto; (d) washing the solid support-first specific bindingmember-analyte-second specific binding member complexes with a secondwash buffer to remove any second specific binding member not bound tothe analyte bound to the first specific binding member thereby producingwashed solid support complexes; (e) adding to the washed solid supportcomplexes a signal generating substrate and a colorant, wherein thesignal generating compound and the signal generating substrate produce adetectable signal; (f) spatially segregating at least a portion of thewashed solid support complexes into a plurality of separate locations,wherein the plurality of separation locations comprises a plurality ofreaction vessels and wherein the washed solid support complexes arepresent in an aqueous phase; (g) covering the plurality of reactionvessels with a sealant, wherein the aqueous phase is displaced by thesealant in the reaction chamber; and (h) detecting the presence orabsence of the detectable signal using a digital counting device,wherein detection of the presence of the detectable signal indicates thepresence of a single molecule of analyte in the sample.

Clause 2. The method of clause 1, wherein the signal-generating digitalassay is a fluorescent digital immunoassay.

Clause 3. A fluorescent digital immunoassay for determining the presenceor absence of a single molecule of an analyte in a fluid sample, themethod comprising: (a) contacting the fluid sample containing orsuspected of containing the analyte with a plurality of solid supportsto create a mixture, wherein each solid support comprises one or morefirst specific binding member capable of binding to the analyte therebyproducing solid support-first specific binding member-analyte complexes;(b) washing the solid support-first specific binding member-analytecomplexes in the mixture with a first wash buffer to remove any solidsupport-first specific binding member not bound to the analyte therebyproducing a washed mixture; (c) adding to the washed mixture one or moresecond specific binding members capable of binding to the analytethereby producing solid support-first specific bindingmember-analyte-second specific binding member complexes, wherein thesecond specific binding member comprises a signal generating compoundattached thereto; (d) washing the solid support-first specific bindingmember-analyte-second specific binding member complexes with a secondwash buffer to remove any second specific binding member not bound tothe analyte bound to the first specific binding member thereby producingwashed solid support complexes; (e) adding to the washed solid supportcomplexes a signal generating substrate and a colorant, wherein thesignal generating compound and the signal generating substrate produce adetectable signal; (f) spatially segregating at least a portion of thewashed solid support complexes into a plurality of separate locations,wherein the plurality of separation locations comprises a plurality ofreaction vessels and wherein the washed solid support complexes arepresent in an aqueous phase; (g) covering the plurality of reactionvessels with a sealant, wherein the aqueous phase is displaced by thesealant in the reaction chamber; and (h) detecting the presence orabsence of the detectable signal using a digital counting device,wherein detection of the presence of the detectable signal indicates thepresence of a single molecule of analyte in the sample.

Clause 4. The method of any one of clauses 1 to 3, wherein the colorantis added simultaneously with the signal generating substrate.

Clause 5. The method of any one of clauses 1 to 3, wherein the colorantis added to the washed solid support complexes before the signalgenerating substrate.

Clause 6. The method of any one of clauses 1 to 3, wherein the colorantis added to the washed solid support complexes after the signalgenerating substrate.

Clause 7. A signal-generating digital assay for determining the presenceor absence of a single molecule of an analyte in a fluid sample, themethod comprising: (a) contacting the fluid sample containing orsuspected of containing the analyte with a plurality of solid supportsto create a mixture, wherein each solid support comprises one or morefirst specific binding member capable of binding to the analyte therebyproducing solid support-first specific binding member-analyte complexes;(b) washing the solid support-first specific binding member-analytecomplexes in the mixture with a first wash buffer to remove any solidsupport-first specific binding member not bound to the analyte therebyproducing a washed mixture; (c) adding to the washed mixture one or moresecond specific binding members capable of binding to the analytethereby producing solid support-first specific bindingmember-analyte-second specific binding member complexes, wherein thesecond specific binding member comprises a signal generating compoundattached thereto; (d) washing the solid support-first specific bindingmember-analyte-second specific binding member complexes with a secondwash buffer to remove any second specific binding member not bound tothe analyte bound to the first specific binding member thereby producingwashed solid support complexes; (e) adding to the washed solid supportcomplexes a signal generating substrate, wherein the signal generatingcompound and the signal generating substrate produce a detectablesignal; (f) spatially segregating at least a portion of the washed solidsupport complexes into a plurality of separate locations, wherein theplurality of separation locations comprises a plurality of reactionvessels and wherein the washed solid support complexes are present in anaqueous phase; (g) covering the plurality of reaction vessels with asealant, wherein the aqueous phase is displaced by the sealant in thereaction chamber; (h) adding a colorant to the displaced aqueous phase;and (i) detecting the presence or absence of the detectable signal usinga digital counting device, wherein detection of the presence of thedetectable signal indicates the presence of a single molecule of analytein the sample.

Clause 8. A signal-generating digital assay for determining the presenceor absence of a single molecule of an analyte in a fluid sample, themethod comprising:

(a) contacting the fluid sample containing or suspected of containingthe analyte with a plurality of solid supports to create a mixture,wherein each solid support comprises one or more first specific bindingmember capable of binding to the analyte thereby producing solidsupport-first specific binding member-analyte complexes; (b) washing thesolid support-first specific binding member-analyte complexes in themixture with a first wash buffer to remove any solid support-firstspecific binding member not bound to the analyte thereby producing awashed mixture; (c) adding to the washed mixture one or more secondspecific binding members capable of binding to the analyte therebyproducing solid support-first specific binding member-analyte-secondspecific binding member complexes, wherein the second specific bindingmember comprises a signal generating compound attached thereto; (d)washing the solid support-first specific binding member-analyte-secondspecific binding member complexes with a second wash buffer to removeany second specific binding member not bound to the analyte bound to thefirst specific binding member thereby producing washed solid supportcomplexes; (e) adding to the washed solid support complexes a signalgenerating substrate, wherein the signal generating compound and thesignal generating substrate produce a detectable signal; (f) spatiallysegregating at least a portion of the washed solid support complexesinto a plurality of separate locations, wherein the plurality ofseparation locations comprises a plurality of reaction vessels andwherein the washed solid support complexes are present in an aqueousphase; (g) covering the plurality of reaction vessels with a sealant,wherein the aqueous phase is displaced by the sealant in the reactionchamber; (h) adding a colorant to the sealant; and (i) detecting thepresence or absence of the detectable signal using a digital countingdevice, wherein detection of the presence of the detectable signalindicates the presence of a single molecule of analyte in the sample.

Clause 9. The method of clause 7 or 8, wherein the signal-generatingdigital assay is a fluorescent digital immunoassay.

Clause 10. A fluorescent digital immunoassay for determining thepresence or absence of a single molecule of an analyte in a fluidsample, the method comprising: (a) contacting the fluid samplecontaining or suspected of containing the analyte with a plurality ofsolid supports to create a mixture, wherein each solid support comprisesone or more first specific binding member capable of binding to theanalyte thereby producing solid support-first specific bindingmember-analyte complexes; (b) washing the solid support-first specificbinding member-analyte complexes in the mixture with a first wash bufferto remove any solid support-first specific binding member not bound tothe analyte thereby producing a washed mixture; (c) adding to the washedmixture one or more second specific binding members capable of bindingto the analyte thereby producing solid support-first specific bindingmember-analyte-second specific binding member complexes, wherein thesecond specific binding member comprises a signal generating compoundattached thereto; (d) washing the solid support-first specific bindingmember-analyte-second specific binding member complexes with a secondwash buffer to remove any second specific binding member not bound tothe analyte bound to the first specific binding member thereby producingwashed solid support complexes; (e) adding to the washed solid supportcomplexes a signal generating substrate, wherein the signal generatingcompound and the signal generating substrate produce a detectablesignal; (f) spatially segregating at least a portion of the washed solidsupport complexes into a plurality of separate locations, wherein theplurality of separation locations comprises a plurality of reactionvessels and wherein the washed solid support complexes are present in anaqueous phase; (g) covering the plurality of reaction vessels with asealant, wherein the aqueous phase is displaced by the sealant in thereaction chamber; (h) adding a colorant to the displaced aqueous phase;and (i) detecting the presence or absence of the detectable signal usinga digital counting device, wherein detection of the presence of thedetectable signal indicates the presence of a single molecule of analytein the sample.

Clause 11. A fluorescent digital immunoassay for determining thepresence or absence of a single molecule of an analyte in a fluidsample, the method comprising: (a) contacting the fluid samplecontaining or suspected of containing the analyte with a plurality ofsolid supports to create a mixture, wherein each solid support comprisesone or more first specific binding member capable of binding to theanalyte thereby producing solid support-first specific bindingmember-analyte complexes; (b) washing the solid support-first specificbinding member-analyte complexes in the mixture with a first wash bufferto remove any solid support-first specific binding member not bound tothe analyte thereby producing a washed mixture; (c) adding to the washedmixture one or more second specific binding members capable of bindingto the analyte thereby producing solid support-first specific bindingmember-analyte-second specific binding member complexes, wherein thesecond specific binding member comprises a signal generating compoundattached thereto; (d) washing the solid support-first specific bindingmember-analyte-second specific binding member complexes with a secondwash buffer to remove any second specific binding member not bound tothe analyte bound to the first specific binding member thereby producingwashed solid support complexes; (e) adding to the washed solid supportcomplexes a signal generating substrate, wherein the signal generatingcompound and the signal generating substrate produce a detectablesignal; (f) spatially segregating at least a portion of the washed solidsupport complexes into a plurality of separate locations, wherein theplurality of separation locations comprises a plurality of reactionvessels and wherein the washed solid support complexes are present in anaqueous phase; (g) covering the plurality of reaction vessels with asealant, wherein the aqueous phase is displaced by the sealant in thereaction chamber; (h) adding a colorant to the sealant; and (i)detecting the presence or absence of the detectable signal using adigital counting device, wherein detection of the presence of thedetectable signal indicates the presence of a single molecule of analytein the sample.

Clause 12. The method of any one of clauses 1 to 11, wherein thecolorant is a black colorant.

Clause 13. The method of any one of clauses 1 to 12, wherein thecolorant is a pigment-based composition.

Clause 14. The method of any one of clauses 1 to 13, wherein thecolorant is one of India Ink, Acid Black 2, Acid Orange 7, Direct Blue14, or a combination thereof.

Clause 15. The method of any one of clauses 2 to 6 and 9 to 14, whereinthe fluorescent digital immunoassay has reduced background fluorescence.

Clause 16. The method of any one of clauses 1 to 15, wherein the one ormore second specific binding members are added simultaneously orsequentially to the one or more first specific binding members.

Clause 17. The method of any one of clauses 1 to 16, wherein the signalgenerating compound is alkaline phosphatase or β-galactosidase.

Clause 18. The method of any one of clauses 1 to 17, wherein the signalgenerating substrate is a fluorescent substrate for the signalgenerating compound.

Clause 19. The method of any one of clauses 1 to 18, wherein the signalgenerating substrate is 4-methylumbelliferyl phosphate (MUP) orfluorescein diphosphate (FDP).

Clause 20. The method of any one of clauses 1 to 19, wherein thedetectable signal is detected using a fluorescence microscope to acquirefluorescence images.

Clause 21. The method of any one of clauses 1 to 20, wherein the sealanthas a density that is heavier than the aqueous phase.

Clause 22. The method of any one of clauses 1 to 21, wherein the sealantis an oil.

Clause 23. The method of clause 22, wherein the oil is a heavyfluorinated oil.

Clause 24. The method of clause 23, wherein the heavy fluorinated oil isFC-40.

Clause 25. The method of any one of clauses 1 to 24, wherein theplurality of reaction vessels is a nanowell array.

Clause 26. The method of any one of clauses 1 to 25, further comprisingin step (e) an inhibitor of signal generating compound.

Clause 27. The method of clause 26, wherein the inhibitor is levamisole.

Clause 28. The method of any one of clauses 1 to 27, further comprisingincubating the mixture for a period of time before adding to the mixtureone or more second specific binding members or after adding to themixture one or more second specific binding members, wherein the periodof time is an incubation period and is about 40 minute to about 300minutes.

Clause 29. The method of any one of clauses 1 to 28, wherein the fluidsample is serum, plasma or a whole blood sample.

Clause 30. The method of any one of clauses 1 to 29, further comprisingcontacting the fluid sample with one or more detergents, a surfactant, anonpolar solvent, sonication, heating, or combination thereof, prior tocontacting the fluid sample to the plurality of solid supports.

Clause 31. The method of any one of clauses 1 to 30, wherein the solidsupport is a magnetic solid support.

Clause 32. The method of any one of clauses 1 to 31, wherein the aqueousphase is displaced by the sealant in step (g) by tilting the pluralityof reaction vessels.

Clause 33. A method of any one of clauses 1 to 32, wherein the analyteis a biological molecule.

Clause 34. The method of clause 33, wherein the biological molecule is aprotein, a peptide, a DNA molecule, a RNA molecule, a sugar, or a lipid.

Clause 35. A method for reducing fluorescence background noise in asignal-generating digital assay used to detect an analyte in a sample,the method comprising: (a) contacting a fluid sample containing orsuspected of containing the analyte with a plurality of solid supportsto create a mixture, wherein each solid support comprises one or morefirst specific binding member capable of binding to the analyte therebyproducing solid support-first specific binding member-analyte complexes;(b) washing the solid support-first specific binding member-analytecomplexes in the mixture with a first wash buffer to remove any solidsupport-first specific binding member not bound to the analyte therebyproducing a washed mixture; (c) adding to the washed mixture one or moresecond specific binding members capable of binding to the analytethereby producing solid support-first specific bindingmember-analyte-second specific binding member complexes, wherein thesecond specific binding member comprises a signal generating compoundattached thereto; (d) washing the solid support-first specific bindingmember-analyte-second specific binding member complexes with a secondwash buffer to remove any second specific binding member not bound tothe analyte bound to the first specific binding member thereby producingwashed solid support complexes; (e) adding to the washed solid supportcomplexes a signal generating substrate, wherein the signal generatingcompound and the signal generating substrate produce a detectablesignal; (f) spatially segregating at least a portion of the washed solidsupport complexes into a plurality of separate locations, wherein theplurality of separation locations comprises a plurality of reactionvessels and wherein the washed solid support complexes are present in anaqueous phase; (g) covering the plurality of reaction vessels with asealant, wherein the aqueous phase is displaced by the sealant in thereaction chamber; and (h) detecting the presence or absence of thedetectable signal using a digital counting device, wherein detection ofthe presence of the detectable signal indicates the presence of a singlemolecule of analyte in the sample, wherein the digital counting devicecomprises a black device.

Clause 36. The method of clause 35, wherein the signal-generatingdigital assay is a fluorescent digital immunoassay.

Clause 37. A method for reducing fluorescence background noise in afluorescent digital immunoassay used to detect an analyte in a sample,the method comprising: (a) contacting a fluid sample containing orsuspected of containing the analyte with a plurality of solid supportsto create a mixture, wherein each solid support comprises one or morefirst specific binding member capable of binding to the analyte therebyproducing solid support-first specific binding member-analyte complexes;(b) washing the solid support-first specific binding member-analytecomplexes in the mixture with a first wash buffer to remove any solidsupport-first specific binding member not bound to the analyte therebyproducing a washed mixture; (c) adding to the washed mixture one or moresecond specific binding members capable of binding to the analytethereby producing solid support-first specific bindingmember-analyte-second specific binding member complexes, wherein thesecond specific binding member comprises a signal generating compoundattached thereto; (d) washing the solid support-first specific bindingmember-analyte-second specific binding member complexes with a secondwash buffer to remove any second specific binding member not bound tothe analyte bound to the first specific binding member thereby producingwashed solid support complexes; (e) adding to the washed solid supportcomplexes a signal generating substrate, wherein the signal generatingcompound and the signal generating substrate produce a detectablesignal; (f) spatially segregating at least a portion of the washed solidsupport complexes into a plurality of separate locations, wherein theplurality of separation locations comprises a plurality of reactionvessels and wherein the washed solid support complexes are present in anaqueous phase; (g) covering the plurality of reaction vessels with asealant, wherein the aqueous phase is displaced by the sealant in thereaction chamber; and (h) detecting the presence or absence of thedetectable signal using a digital counting device, wherein detection ofthe presence of the detectable signal indicates the presence of a singlemolecule of analyte in the sample, wherein the digital counting devicecomprises a black device.

Clause 38. The method of any one of clauses 35 to 37, wherein the blackdevice comprises a solid phase material comprising carbon black or ablack film sheet attached to a transparent device.

Clause 39. The method of clause 38, wherein the solid phase material isCYTOP, cyclic olefin polymers (COP), or polydimethylsiloxane (PDMS).

Clause 40. The method of any one of clauses 35 to 39, further comprisingadding a colorant to the washed solid support complexes simultaneouslywith the signal generating substrate, adding a colorant to the washedsolid support complexes before the signal generating substrate, adding acolorant to the washed solid support complexes after the signalgenerating substrate, adding a colorant to the displaced aqueous phase,or adding a colorant to the sealant.

Clause 41. The method of clause 40, wherein the colorant is apigment-based composition.

Clause 42. The method of clause 40 or 41, wherein the colorant is one ofIndia Ink, Acid Black 2, Acid Orange 7, Direct Blue 14, or a combinationthereof.

Clause 43. The method of clause 42, wherein the colorant is acombination of Acid Orange 7 and Direct Blue 14.

Clause 44. The method of any one of clauses 35 to 43, wherein the one ormore second specific binding members are added simultaneously orsequentially to the one or more first specific binding members.

Clause 45. The method of any one of clauses 35 to 44, wherein the signalgenerating compound is alkaline phosphatase or β-galactosidase.

Clause 46. The method of any one of clauses 35 to 45, wherein the signalgenerating substrate is a fluorescent substrate for the signalgenerating compound.

Clause 47. The method of any one of clauses 35 to 46, wherein the signalgenerating substrate is 4-methylumbelliferyl phosphate (MUP) orfluorescein diphosphate (FDP).

Clause 48. The method of any one of clauses 35 to 47, wherein thedetectable signal is detected using a fluorescence microscope to acquirefluorescence images.

Clause 49. The method of any one of clauses 35 to 48, wherein thesealant has a density that is heavier than the aqueous phase.

Clause 50. The method of any one of clauses 35 to 49, wherein thesealant is an oil.

Clause 51. The method of clause 50, wherein the oil is a heavyfluorinated oil.

Clause 52. The method of clause 51, wherein the heavy fluorinated oil isFC-40.

Clause 53. The method of any one of clauses 35 to 52, wherein theplurality of reaction vessels is a nanowell array.

Clause 54. The method of any one of clauses 35 to 53, further comprisingin step (e) an inhibitor of signal generating compound.

Clause 55. The method of clause 54, wherein the inhibitor is levamisole.

Clause 56. The method of any one of clauses 35 to 55, further comprisingincubating the mixture for a period of time before adding to the mixtureone or more second specific binding members or after adding to themixture one or more second specific binding members, wherein the periodof time is an incubation period and is about 40 minute to about 300minutes.

Clause 57. The method of any one of clauses 35 to 56, wherein the fluidsample is serum, plasma or a whole blood sample.

Clause 58. The method of any one of clauses 35 to 57, further comprisingcontacting the fluid sample with one or more detergents, a surfactant, anonpolar solvent, sonication, heating, or combination thereof, prior tocontacting the fluid sample to the plurality of solid supports.

Clause 59. The method of any one of clauses 35 to 58, wherein the solidsupport is a magnetic solid support.

Clause 60. The method of any one of clauses 35 to 59, wherein theaqueous phase is displaced by the sealant in step (g) by tilting theplurality of reaction vessels.

Clause 61. The method of any one of clauses 35 to 60, wherein theanalyte is a biological molecule.

Clause 62. The method of clause 61, wherein the biological molecule is aprotein, a peptide, a DNA molecule, a RNA molecule, a sugar, or a lipid.

Clause 63. The method of any one of clauses 36 to 62, wherein thefluorescent digital immunoassay includes a femto-liter droplet array.

1.-34. (canceled)
 35. A method for reducing fluorescence backgroundnoise in a signal-generating digital assay used to detect an analyte ina sample, the method comprising: (a) contacting a fluid samplecontaining or suspected of containing the analyte with a plurality ofsolid supports to create a mixture, wherein each solid support comprisesone or more first specific binding member capable of binding to theanalyte thereby producing solid support-first specific bindingmember-analyte complexes; (b) washing the solid support-first specificbinding member-analyte complexes in the mixture with a first wash bufferto remove any solid support-first specific binding member not bound tothe analyte thereby producing a washed mixture; (c) adding to the washedmixture one or more second specific binding members capable of bindingto the analyte thereby producing solid support-first specific bindingmember-analyte-second specific binding member complexes, wherein thesecond specific binding member comprises a signal generating compoundattached thereto; (d) washing the solid support-first specific bindingmember-analyte-second specific binding member complexes with a secondwash buffer to remove any second specific binding member not bound tothe analyte bound to the first specific binding member thereby producingwashed solid support complexes; (e) adding to the washed solid supportcomplexes a signal generating substrate, wherein the signal generatingcompound and the signal generating substrate produce a detectablesignal; (f) spatially segregating at least a portion of the washed solidsupport complexes into a plurality of separate locations, wherein theplurality of separation locations comprises a plurality of reactionvessels and wherein the washed solid support complexes are present in anaqueous phase; (g) covering the plurality of reaction vessels with asealant, wherein the aqueous phase is displaced by the sealant in thereaction chamber; and (h) detecting the presence or absence of thedetectable signal using a digital counting device, wherein detection ofthe presence of the detectable signal indicates the presence of a singlemolecule of analyte in the sample, wherein the digital counting devicecomprises a black device.
 36. The method of claim 35, wherein thesignal-generating digital assay is a fluorescent digital immunoassay.37. A method for reducing fluorescence background noise in a fluorescentdigital immunoassay used to detect an analyte in a sample, the methodcomprising: (a) contacting a fluid sample containing or suspected ofcontaining the analyte with a plurality of solid supports to create amixture, wherein each solid support comprises one or more first specificbinding member capable of binding to the analyte thereby producing solidsupport-first specific binding member-analyte complexes; (b) washing thesolid support-first specific binding member-analyte complexes in themixture with a first wash buffer to remove any solid support-firstspecific binding member not bound to the analyte thereby producing awashed mixture; (c) adding to the washed mixture one or more secondspecific binding members capable of binding to the analyte therebyproducing solid support-first specific binding member-analyte-secondspecific binding member complexes, wherein the second specific bindingmember comprises a signal generating compound attached thereto; (d)washing the solid support-first specific binding member-analyte-secondspecific binding member complexes with a second wash buffer to removeany second specific binding member not bound to the analyte bound to thefirst specific binding member thereby producing washed solid supportcomplexes; (e) adding to the washed solid support complexes a signalgenerating substrate, wherein the signal generating compound and thesignal generating substrate produce a detectable signal; (f) spatiallysegregating at least a portion of the washed solid support complexesinto a plurality of separate locations, wherein the plurality ofseparation locations comprises a plurality of reaction vessels andwherein the washed solid support complexes are present in an aqueousphase; (g) covering the plurality of reaction vessels with a sealant,wherein the aqueous phase is displaced by the sealant in the reactionchamber; and (h) detecting the presence or absence of the detectablesignal using a digital counting device, wherein detection of thepresence of the detectable signal indicates the presence of a singlemolecule of analyte in the sample, wherein the digital counting devicecomprises a black device.
 38. The method of claim 35, wherein the blackdevice comprises a solid phase material comprising carbon black or ablack film sheet attached to a transparent device.
 39. The method ofclaim 38, wherein the solid phase material is CYTOP, cyclic olefinpolymers (COP), or polydimethylsiloxane (PDMS).
 40. The method of claim35, further comprising adding a colorant to the washed solid supportcomplexes simultaneously with the signal generating substrate, adding acolorant to the washed solid support complexes before the signalgenerating substrate, adding a colorant to the washed solid supportcomplexes after the signal generating substrate, adding a colorant tothe displaced aqueous phase, or adding a colorant to the sealant. 41.The method of claim 40, wherein the colorant is a pigment-basedcomposition.
 42. The method of claim 40, wherein the colorant is one ofIndia Ink, Acid Black 2, Acid Orange 7, Direct Blue 14, or a combinationthereof.
 43. The method of claim 42, wherein the colorant is acombination of Acid Orange 7 and Direct Blue
 14. 44. The method of claim35, wherein the one or more second specific binding members are addedsimultaneously or sequentially to the one or more first specific bindingmembers.
 45. The method of claim 35, wherein the signal generatingcompound is alkaline phosphatase or β-galactosidase.
 46. The method ofclaim 35, wherein the signal generating substrate is a fluorescentsubstrate for the signal generating compound.
 47. The method of claim35, wherein the signal generating substrate is 4-methylumbelliferylphosphate (MUP) or fluorescein diphosphate (FDP).
 48. The method ofclaim 35, wherein the detectable signal is detected using a fluorescencemicroscope to acquire fluorescence images.
 49. The method of claim 35,wherein the sealant has a density that is heavier than the aqueousphase.
 50. The method of claim 35, wherein the sealant is an oil. 51.The method of claim 50, wherein the oil is a heavy fluorinated oil. 52.The method of claim 51, wherein the heavy fluorinated oil is FC-40. 53.The method of claim 35, wherein the plurality of reaction vessels is ananowell array.
 54. The method of claim 35, further comprising in step(e) adding an inhibitor of signal generating compound.
 55. The method ofclaim 54, wherein the inhibitor is levamisole.
 56. The method of claim35, further comprising incubating the mixture for a period of timebefore adding to the mixture one or more second specific binding membersor after adding to the mixture one or more second specific bindingmembers, wherein the period of time is an incubation period and is about40 minute to about 300 minutes.
 57. The method of claim 35, wherein thefluid sample is serum, plasma or a whole blood sample.
 58. The method ofclaim 35, further comprising contacting the fluid sample with one ormore detergents, a surfactant, a nonpolar solvent, sonication, heating,or combination thereof, prior to contacting the fluid sample to theplurality of solid supports.
 59. The method of claim 35, wherein thesolid support is a magnetic solid support.
 60. The method of claim 35,wherein the aqueous phase is displaced by the sealant in step (g) bytilting the plurality of reaction vessels.
 61. The method of claim 35,wherein the analyte is a biological molecule.
 62. The method of claim61, wherein the biological molecule is a protein, a peptide, a DNAmolecule, a RNA molecule, a sugar, or a lipid.
 63. The method of claim35, wherein the fluorescent digital immunoassay includes a femto-literdroplet array.